Biomarkers for treatment of neoplastic disorders using androgen-targeted therapies

ABSTRACT

Described herein are methods and compositions for the treatment of prostate cancer in a subject in need thereof. The prostate cancer may be a castration resistant and an androgen receptor antagonist-resistant prostate cancer. The methods may comprise administering to the subject a CYP17-lyase inhibitor of Formula II.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/865,038, filed Aug. 12, 2013, U.S. Provisional Application No.61/990,570, filed on May 8, 2014, and U.S. Provisional Application No.62/002,110, filed on May 22, 2014, which applications are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

Cancer represents a significant burden on human health, accounting foran estimated 13% of all deaths each year. In particular, several commoncancers and diseases are associated with androgen hormone signaling,such as, for example, prostate cancer, breast cancer, ovarian cancer,bladder cancer, pancreatic cancer, and polycystic ovary disease. Forexample, prostate cancer (PCa) is the second most common cancer in men.The majority of prostate cancer deaths are due to the development ofmetastatic disease that is unresponsive to conventional androgendeprivation therapy. Androgen deprivation therapy has been the standardof care in subjects with prostate cancer since the 1940s. Despiteandrogen deprivation, most subjects ultimately experience diseaseprogression. For many years this later phase of the disease was called“hormone insensitive prostate cancer” or “androgen independent prostatecancer.” It has since become clear that the prostate cancer that emergesafter androgen deprivation therapy remains dependent upon androgen. Theprostate cancer cells that have survived have gained the ability toimport low levels of circulating androgens (expressed from adrenalglands), become much more sensitive to these low levels of testosterone,and actually synthesize testosterone within the prostate cancer cellitself. This stage of prostate cancer is now termed “castrationresistant prostate cancer” or CRPC.

Identification of patients that are likely to respond or identificationof those patients that are responding to therapy for prostate cancer isa goal for medical management of this disease. While current clinicalguidelines are focused on symptoms, blood levels of prostate specificantigen (PSA), and imaging studies, other biological markers may beuseful for clinical decision making. There remains a need for biomarkersof the disease and their relationship to identification of efficacy ortoxicity of a therapeutic compound, and biomarkers which could provideinformation regarding identification of patients most likely to respondto therapeutic agents, or to identify patients receiving therapeuticagents who are not responding (either through primary or acquiredresistance mechanisms), or to predict those patients that may developundesirable side effects.

SUMMARY OF THE INVENTION

The biomarkers identified below may be used, for example, to evaluatetreatment options at various points in the course of treatment of apatient. For example, the biomarkers are used in the evaluation oftreatment options at various cancer treatment transition points byanalysis of one ore more biomarkers or a biomarker panel to identify andoptimize therapeutic choices. Biomarkers may also be used to predictunresponsiveness or poor responsiveness to existing anti-cancer therapyand responsiveness to galeterone in the same patient. In one embodiment,biomarker detection is performed and correlated with galeterone efficacyin the treatment of prostate cancer.

A method of treating a disease in a patient in need thereof is provided,comprising: a) determining whether the disease is characterized by analtered form of androgen receptor; and b) if said altered form ofandrogen receptor is present, administering to said subject apharmaceutical composition comprising a therapeutically effective amountof a compound of Formula I,

or a pharmaceutically acceptable salt, N-oxide, active metabolite,prodrug, or solvate thereof; wherein R₁ is H or acetyl; and R₂ isbenzimidazole.

For example, R₁ is acetyl and the compound is a pro-drug of galeterone.

For example, R₁ is H and R₂ is benzamidazole and the compound isgaleterone.

Provided herein is a method of treating a cancer in a patient in needthereof, comprising: a) obtaining a sample from said patient, forexample a sample of circulating tumor cells; b) determining whether atruncated form of androgen receptor is present in the sample, forexample ARV-7 or AR-V567es; and c) if said altered form of androgenreceptor is present, administering to said subject a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula I,

or a pharmaceutically acceptable salt, N-oxide, active metabolite,prodrug, or solvate thereof; wherein R₁ is H or acetyl; and R₂ isbenzimidazole. For example, R₁ is hydrogen, R₂ is benzimidazole and thecompound is galeterone. In some embodiments, the altered form ofandrogen receptor is a truncated AR lacking the ligand-binding domain,for example ARV-7. Said determining may include, for example, use of anantibody capable of binding to a ligand binding domain of the androgenreceptor, such that absence of binding of the antibody results inabsence of signal. Said determining may also include the use of twoantibodies, for example one to the NH₂ terminal of the AR and one to theCOOH terminal (ligand binding domain) of the AR to differentiate orgenerate a ratio of the presence of the NH2 terminal and absence of theCOOH terminal (ligand binding). In a preferred embodiment, the analysisresult is a ratio of antibody detection signals of the NH2 terminal andthe C-terminal in the same subject sample. Said determining may alsoinclude detection of a truncated AR lacking the ligand binding domain bynucleic acid amplification, either quantitative or qualitative, or by agene expression assay. In one embodiment, the detection of the truncatedAR lacking the ligand binding domain is from a patient or subjectsample, for example where the patient/subject sample has been enrichedfor circulating tumor cells. In another embodiment the patient/subjectsample is enriched for circulating DNA. In yet another preferredembodiment, the patient/subject sample is a tumor biopsy or tissuesample.

In some embodiments, the disease is a prostate disease, for exampleprostate cancer. The prostate cancer may be resistant to castration. Insome cases, the subject has undergone castration, for example chemicalcastration or surgical castration, or has undergone androgen receptorantagonist treatment or treatment to reduce nascent androgen production,such as interference with the steroidogenesis pathway, for example aCYP-17 lyase inhibitor, or combination therapy. In some embodiments, thedisease is cancer, such as ovarian, bladder, pancreatic or breastcancer. The cancer may be resistant to an anti-androgen such as anandrogen receptor antagonist, for example to enzalutamide orbicalutamide or ARN-509. The cancer may be resistant to a CYP17-lyaseinhibitor, for example to abiraterone. The cancer may be resistant to ataxane, for example docetaxel or cabazitaxel. In some embodiments, thedisease is androgen dependent.

The patient may be determined to have a mutated androgen receptor, forexample a truncated AR such as AR-V1, AR-V2, AR-V3, AR-V4. AR-V5,AR-V567es, AR-V6, or AR-V7. The mutated AR can carry a point mutationsuch as T877A (T878A), D879G, (D878G), W741C, W741L, M749L, R629Q,G142V, P533S, T575A, H874Y, or F876L.

Also provided herein are methods of optimizing therapy of a disease in apatient in need thereof, comprising: identifying a patient undergoingtreatment of a disease using a treatment regimen, wherein said treatmentregimen comprises administration of a compound of Formula I:

or a pharmaceutically acceptable salt, N-oxide, active metabolite,prodrug, or solvate thereof; wherein R₁ is H or acetyl; and R₂ isbenzimidazole;determining the status of at least one biomarker; and based on thedetermination of said biomarker, maintaining or modifying the therapyregimen. In a preferred embodiment, R₁ is hydrogen and R₂ isbenzamidazole.

For example, the biomarker is the expression level or function of anandrogen receptor, for example a wild-type or mutated AR. The mutated ARcan be a splice variant and/or truncated AR, including AR-V1, AR-V2,AR-V3, AR-V4. AR-V5, AR-V567es, AR-V6, or AR-V7. Mutated AR can be ARwith point mutations including, but not limited to, T877A (T878A), D879G(D878G), W741C, W741L, M749L, R629Q, G142V, P533S, T575A, H874Y, F876L.

In some embodiments, the disease is a prostate disease, for exampleprostate cancer. The prostate cancer may be resistant to castration. Insome embodiments, the disease is cancer, such as ovarian, bladder,pancreatic or breast cancer. The cancer may be resistant to ananti-androgen such as an androgen receptor antagonist, for example toenzalutamide or bicalutamide or ARN-509. The cancer may be resistant totaxanes, for example docetaxel or cabazitaxel. In some cases, thesubject has undergone castration or has undergone androgen receptorantagonist treatment, or both. In some embodiments, the disease is anandrogen dependent disease.

The biomarker may also be a decrease in the number of circulating tumorcells (CTCs), for example wherein the number of circulating tumor cellsis determined after at least 1 week of galeterone therapy. Suitablebiomarkers include, but are not limited to: an increase in apoptoticCTCs; a decrease in PSA or a reduction in PSA doubling time; an increasein PSMA expression; a reduction in the tumor ¹⁸F-DHT-PET signal; atissue biopsy based test, for example ProMark; the presence orexpression of a proteosome degradation pathway member; a 5-kallikreinpanel; the pre- and post-treatment testosterone blood level; a change inthe level of at least one steroid after initiation of the treatmentregimen; a metabolic marker, for example in the level of a P450 enzyme;a mutation or variant of a CYP17 protein; determination of a CTLA-4blockade; a prostate health index; the presence or level of PCA3; theprostate core mitomic test; the presence of a mutation in a cell cycleprogression gene; the level of hemoglobin, lactate dehydrogenase, oralkaline phosphatase, as determined by a serologic test; and resistanceto chemotherapy (including enzalutamide or abiraterone, bicalutamide orARN-509).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts androgen receptor (AR) and alternative splice variantsthereof.

FIG. 2 shows that galeterone downregulates full-length and splicevariant AR and reduces cell proliferation in a CWR22rv1 cell line.

FIG. 3 shows that galeterone overcomes abiraterone and enzalutamideresistance due to AR splice variants. Galeterone, but not enzalutamide,reduces full-length and splice variant AR-V7 protein.

FIG. 4 shows that galeterone reduces both full-length and AR-V7 proteinsin a CWR22rv1 cell line.

FIG. 5 shows that galeterone reduces AR-V7 in DU145 cells transfectedwith AR-V7 splice variant.

FIG. 6 shows that galeterone reduces full-length and splice variantAR-V7 with 72 hour exposure.

FIG. 7 depicts proliferation of castration resistant and enzalutamideresistant cell lines.

FIG. 8 depicts response of castration resistant and enzalutamideresistant cell lines to galeterone.

FIG. 9A depicts an Androgen Receptor (AR) and Prostate Specific Antigen(PSA) western blot of LNCaP and enzalutamide resistant LNCaP cells.

FIG. 9B depicts the effect of galeterone on AR and PSA protein levels inenzalutamide responsive and enzalutamide resistant LNCaP cells.

FIG. 10 depicts androgen receptor localization in cells treated with asynthetic androgen in the presence or absence of enzalutamide.

FIG. 11 depicts androgen receptor localization in a CPRC cell linetreated with a synthetic androgen in the presence or absence ofenzalutamide or galeterone.

FIG. 12 depicts androgen receptor localization in an enzalutamideresistant cell line treated with a synthetic androgen in the presence orabsence of enzalutamide or galeterone.

FIG. 13 depicts androgen receptor localization in an enzalutamideresistant cell line treated with a synthetic androgen in the presence orabsence of enzalutamide or galeterone.

FIG. 14 depicts AR luciferase reporter activity in cell lines treatedwith enzalutamide or galeterone.

FIG. 15A shows the design of an immunofluorescence experiment tovisualize nuclear localization of test compounds used.

FIG. 15B describes the results of the experiment described in FIG. 15Aand shows that galeterone, but not enzalutamide, reduces AR nucleartranslocation.

FIG. 16 shows that castration resistant tumors which express AR-V7respond to galeterone. AR-V7 was detected in LuCaP136 castrationresistant xenograft tumors using RT-PCR.

FIG. 17 shows that galeterone downregulates androgen receptors carryingthe AR-T878A mutation.

FIG. 18 shows the results of a binding assay of galeterone to wild typeAR and AR point mutants.

FIG. 19 shows that galeterone reduces AR-dependent gene expression incells with wild-type AR and cells with AR point mutations. These cellsalso express AR receptors with F876L mutation.

FIG. 20 shows decrease in PSA induced by PSA in CRPC and enzalutamideresistant cell lines.

FIG. 21 depicts a scheme illustrating the detection of AR variants inwhich the C-terminal domain has been lost.

FIG. 22 shows the results of a study in which patients with C-terminalAR loss (4/4) treated with galeterone show maximal PSA response (>50%).

DETAILED DESCRIPTION OF THE INVENTION Definitions

A “subject” as used herein refers to a patient or subject in a clinicaltrial and more broadly a biological entity containing expressed geneticmaterials. Tissues (including biopsied materials), cells and theirprogeny from a subject obtained in vivo or cultured in vitro are alsoencompassed.

By “sample” is meant a fluid, solid, or tissue removed from a subjectand includes whole blood, serum, plasma, tissue, semen, cell, biopsy,mucous, feces, bone, teeth, nasal or throat or cheek swab, urine, skin,tears, organ biopsy (liver, kidney, colon, lung, pancreas), tumorbiopsy, or tumor tissue, circulating tumor cells, exosomes from theprimary tumor or metastatic tissue. The sample may also include aportion of the collected fluid, solid, or tissue from a subject, forexample a circulating tumor cell, or an analyte. By “processed sample”is meant the fluid, solid, or tissue from the subject is treated,handled, or managed via laboratory techniques to enrich for an analyte.By “processed whole blood sample” is meant that the whole blood sampleis processed through in vitro laboratory techniques to analyze acomponent of the whole blood sample including cells, DNA, RNA, proteins,peptides, or an analyte as described below; for example, cells found ina whole blood sample may be enriched, washed, and analyzed separately;more specifically, circulating tumor cells may be enriched from a wholeblood sample and analyzed for a biomarker or biomarkers, and thesebiomarkers may include a mutated form of androgen receptor. Anotherexample is to enrich a whole blood sample for DNA (for examplecirculating DNA or tumor cell DNA) that can be analyzed for a biomarkeror biomarkers and these biomarkers may include a mutated form ofandrogen receptor.

By “analyte” is meant a substance or a constituent of a sample to beanalyzed. Exemplary analytes include one or more species of one or moreof the following: a protein, a peptide, a polypeptide, an amino acid, anucleic acid, an oligonucleotide, mRNA, RNA, microRNA, long non-codingRNA, DNA, circulating DNA, cDNA, an antibody, a carbohydrate, apolysaccharide, glucose, a lipid, a gas (e.g., oxygen or carbondioxide), an electrolyte (e.g., sodium, potassium, chloride,bicarbonate, BUN, magnesium, phosphate, calcium, ammonia, lactate, zinc,citrate), a lipoprotein, cholesterol, a fatty acid, a glycoprotein, aproteoglycan, a lipopolysaccharide, a cell surface marker (e.g., CD3,CD4, CD8, IL2R, or CD35), a tumor marker (BCL-2, Ki-67, ERK5), prostatespecific antigen (PSA), a cytoplasmic marker, a therapeutic agent, ametabolite of a therapeutic agent, a cell (e.g., a whole cell, a tumorcell, a circulating tumor cell, a stem cell, a white blood cell, a Tcell (e.g., displaying CD3, CD4, CD8, IL2R, CD35, or other surfacemarkers), or another cell identified with one or more specific markers).As used herein, the term “small molecule” refers to a drug, medication,medicament, or other chemically synthesized compound that iscontemplated for human therapeutic use. As used herein, the term“biologic” refers to a substance derived from a biological source, notsynthesized and that is contemplated for human therapeutic use. Abiomarker is a biological substance that can be used as an indicator ofa particular disease state or particular physiological state of anorganism, generally a biomarker is a protein or other native compoundmeasured in bodily fluid whose concentration reflects the presence orseverity or staging of a disease state or dysfunction, can be used tomonitor therapeutic progress of treatment of a disease or disorder ordysfunction, or can be used as a surrogate measure of clinical outcomeor progression. As used herein, the term “metabolic biomarker” refers toa substance, molecule, or compound that is synthesized or biologicallyderived that is used to determine the status of a patient or subject'sliver or kidney function. As used herein, the term “genotyping” refersto the ability to determine genetic differences in specific genes thatmay or may not affect the phenotype of the specific gene. As usedherein, the term “phenotype” refers to the resultant biologicalexpression, (metabolic or physiological) of the protein set by thegenotype. As used herein, the term “gene expression profiling” refers tothe ability to determine the rate or amount of the production of a geneproduct or the activity of gene transcription in a specific tissue, in atemporal or spatial manner. As used herein, the term “proteomicanalysis” refers to a protein pattern or array to identify keydifferences in proteins or peptides in normal and diseased tissues.Additional exemplary analytes are described herein. The term analytefurther includes components of a sample that are a direct product of abiochemical means of amplification of the initial target analyte, suchas the product of a nucleic acid amplification reaction.

Combination Therapy: The term “combination therapy”, as used herein,refers to those situations in which two or more different pharmaceuticalagents are administered in overlapping regimens so that the subject issimultaneously exposed to two or more agents or administered in temporalregimens so that the subject is exposed to two or more agents insequence.

Dosing Regimen: A “dosing regimen”, as that term is used herein, refersto a set of unit doses (typically more than one) that are administeredindividually separated by periods of time. The recommended set of doses(i.e., amounts, timing, route of administration, etc.) for a particularpharmaceutical agent constitutes its dosing regimen.

Initiation: As used herein, the term “initiation” when applied to adosing regimen can be used to refer to a first administration of apharmaceutical agent to a subject who has not previously received thepharmaceutical agent. Alternatively or additionally, the term“initiation” can be used to refer to administration of a particular unitdose of a pharmaceutical agent during therapy of a subject.

Pharmaceutical agent: As used herein, the phrase “pharmaceutical agent”refers to any agent that, when administered to a subject, has atherapeutic effect and/or elicits a desired biological and/orpharmacological effect.

Pharmaceutically acceptable ester: As used herein, the term“pharmaceutically acceptable ester” refers to esters which hydrolyze invivo and include those that break down readily in the human body toleave the parent compound or a salt thereof.

Therapeutically effective amount: The term “therapeutically effectiveamount” of a pharmaceutical agent or combination of agents is intendedto refer to an amount of agent(s) which confers a therapeutic effect onthe treated subject, at a reasonable benefit/risk ratio applicable toany medical treatment. The therapeutic effect may be objective (i.e.,measurable by some test or marker) or subjective (i.e., subject gives anindication of or feels an effect). A therapeutically effective amount iscommonly administered in a dosing regimen that may comprise multipleunit doses. For any particular pharmaceutical agent, a therapeuticallyeffective amount (and/or an appropriate unit dose within an effectivedosing regimen) may vary, for example, depending on route ofadministration, on combination with other pharmaceutical agents. Also,the specific therapeutically effective amount (and/or unit dose) for anyparticular subject may depend upon a variety of factors including thedisorder being treated and the severity of the disorder, the activity ofthe specific pharmaceutical agent employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thesubject; the time of administration, route of administration, and/orrate of excretion or metabolism of the specific pharmaceutical agentemployed; the duration of the treatment; and like factors as is wellknown in the medical arts.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a pharmaceutical agent,remedy, or medicament that partially or completely alleviates,ameliorates, relieves, inhibits, delays onset of, reduces severity ofand/or reduces incidence of one or more symptoms or features of aparticular disease, disorder, syndrome and/or condition. Such treatmentmay be of a subject who does not exhibit signs of the relevant disease,disorder and/or condition and/or of a subject who exhibits only earlysigns of the disease, disorder, and/or condition. Alternatively oradditionally, such treatment may be of a subject who exhibits one ormore established signs of the relevant disease, disorder and/orcondition.

As used herein, the term “unresponsiveness” or “poor responsiveness”refers to the lack of change of or minimal change of a patient's orsubject's clinical or medical presentation, prognosis, symptoms,diagnostic indices, features, survival or outcomes after treatment andit further implies and infers that the treatment has not partially orcompletely alleviate, ameliorate, relieve, inhibit, delay onset of,reduce severity of and/or reduce incidence of one or more symptoms orfeatures of a particular disease, disorder, syndrome and/or condition.Unresponsiveness can be used interchangeably with “lack of efficacy”,“lack of response”. Conversely, “response” or “responsiveness” totreatment refers to a change of a patient's clinical or medicalpresentation, prognosis, symptoms, diagnostic indices, features,survival or outcomes after treatment and it further implies and infersthat the treatment has partially or completely alleviate, ameliorate,relieve, inhibit, delay onset of, reduce severity of and/or reduceincidence of one or more symptoms or features of a particular disease,disorder, syndrome and/or condition. Response may be usedinterchangeably with “efficacy” as the capacity of a treatment or remedyfor producing a desired result or effect and is reflective of thequality of the treatment or remedy to produce the intended result. Thus,the biomarkers of the instant invention are used at assisting medicaldecision making. For example, during evaluation of a course oftreatment, a sample is analyzed from the patient for a biomarker or apanel of biomarkers, determining the presence/absence or level of abiomarker or panel of biomarkers and then administering to the patient acompound of formula I or a compound of formula II based on the biomarkeranalysis result.

Unit dose: The term “unit dose” or “dose”, as used herein, refers to adiscrete administration of a pharmaceutical agent, typically in thecontext of a dosing regimen.

Definitions of standard chemistry terms may be found in reference works,including Carey and Sundberg “ADVANCED ORGANIC CHEMISTRY 4^(th) ED.”Vols. A (2000) and B (2001), Plenum Press, New York, hereby incorporatedby reference in its entirety. Unless otherwise indicated, conventionalmethods of mass spectroscopy, NMR, HPLC, protein chemistry,biochemistry, recombinant DNA techniques and pharmacology, within theskill of the art are employed.

Solid dispersion: The term “solid dispersion”, as used herein, refers tocomposition comprising two different components, generally a solidmatrix with a secondary substance (such as an active pharmaceuticalingredient) dispersed within.

Solid matrix: The term “solid matrix” refers to a solid phase in whichmolecules of a second substance (such as an active pharmaceuticalingredient) are embedded or dispersed within.

Methods of Treatment

Once prostate cancer is diagnosed and staged, clinical managementoptions include expectant, regular, or interval management orsurveillance, surgery, radiation therapy, cryosurgery, hormone therapy,chemotherapy, immunotherapy and vaccine treatment. Often the inclusionof age and expected life span and other concomitant health conditionsare considered along with the stage and grade of the tumor in thetreatment options. Since prostate cancer is an androgen-dependentdisease, hormone therapy or androgen deprivation therapy (ADT) orandrogen suppression therapy has the overall goal of reducing the levelsof androgens in the body or to prevent them from reaching the prostatecancer cells (chemical castration). Hormone therapy includes LHRHagonists (Lupron, eligard, goserelin, tripterelin, histrelin); LHRHantagonists (firmagon). Hormone therapy may be used in conjunction withsurgical resection of the tumor, orchietomy (surgical castration), orradiation therapy or radiopharmaceutical (Radium 223 Dichloride, Xofigo(Radium 223 Dichloride). Therapy aimed at reducing the production ofandrogens include abiraterone (a CYP17 inhibitor). Anti-androgen therapyis aimed at inhibiting the androgen receptor and examples are flutamide,bicalutamide, nilutamide, ARN-509 and enzalutamide. Anti-androgentreatment may be combined with orchiectomy or LHRH analogs as first-linehormone therapy. This is called combined androgen blockade (CAB). Otherandrogen suppressing drugs include estrogens and ketoconazole. Thustargeting the androgen receptor signaling pathway has been a drugdevelopment staple and broadly includes CYP17 inhibitors or modulators,antiandrogens, chaperone inhibitors (targeting heat shock proteins,Hsp-27 inhibitor), androgen-receptor modulator (blocking transactivationdomain of the receptor). Vaccine treatment, currently Sipuleucel, isintended to boost the body's immune system to recognize the prostatetumor and lodge an anti-tumor immune response. This form of therapy isnot “off the shelf” as each vaccine is made from the unique white cellsfrom each individual patient after exposing in a lab to prostate acidphosphatase (PAP). Another immunotherapy includes ipilimumab (a CTLA-4antagonist). Castration resistant prostate cancer (CRPC) is the termused for those patients for which androgen deprivation or androgensuppression therapy is no longer effective at slowing the proliferationof the prostate tumor or the metastasis, and it is a stage of thedisease that is associated with primary or acquired resistance totherapy and for which there are few therapeutic options, one being broadcancer chemotherapy (docetaxel and cabazitaxel being examples).

Currently there are available the following compounds for use intreating patients with prostate cancer: Abiraterone Acetate,Bicalutamide, Cabazitaxel, Casodex (Bicalutamide), Degarelix, Docetaxel,Enzalutamide, Goserelin Acetate, Jevtana (Cabazitaxel), LeuprolideAcetate, Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate),Lupron Depot-3 Month (Leuprolide Acetate), Lupron Depot-4 Month(Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Prednisone,Provenge (Sipuleucel-T), Radium 223 Dichloride, Sipuleucel-T, Taxotere(Docetaxel), Viadur (Leuprolide Acetate), Xofigo (Radium 223Dichloride), Xtandi (Enzalutamide), Zoladex (Goserelin Acetate), Zytiga(Abiraterone Acetate).

Galeterone (the compound of Formula II), is being developed as atherapeutic for androgen-sensitive cancers. Galeterone has been shown tobe a potent inhibitor of CYP17 lyase in the steroidogenic pathway anduniquely also to antagonize binding of androgens to the androgenreceptor, and downregulate the androgen receptor. The overall result ofthese effects on the androgen signaling pathway is the inhibition ofprostate cancer growth. (See U.S. Pat. No. 7,875,599, incorporatedherein by reference.)

Methods are provided herein to assist medical decision making inandrogen dependent disease, disorders, syndromes, and conditions as ameans to enhance the assessment and evaluation of, and optimize thepredictive course of, treatment and to optimize the use of a compound offormula I and more preferably formula II. Methods are described andprovided herein to identify patients that are more likely to respond togaleterone therapy or to identify patients who are not responding to thedrug. By prospectively screening patients who are more likely to respondto the drug, galeterone, or a clinical evaluation of therapy options byanalyzing a biomarker or biomarker panel from a patient sample, patientswith prostate cancer will benefit because if selected as more likely torespond to galeterone, they may have a decreased risk of cancerprogression and metastases and overall better outcomes. In addition,diagnostic methods that identify patients who are responding to thedrug, galeterone, will benefit non-responsive patients as these samemethods may identify those patients that could benefit from a switch toa different therapeutic approach far earlier than empiric therapeuticmanagement. Due to its triad of mechanistic advantages, e.g. inhibitingthe production of androgens, antagonizing and down regulating theandrogen receptor activity, galeterone is a potential candidate ofchoice in cases where there is observed PCa therapy resistance. Forexample, if an antiandrogen has lost its ability to inhibit androgenreceptor activity via the ligand binding site on the receptor,galeterone is a choice therapy as it has been shown to function as ananti-proliferative agent in antiandrogen resistant cells. Further, if ataxane has lost its ability to block cell division in a tumor cell orablate proliferation of a tumor, galeterone is a potential candidate ofchoice therapy as it has the potential to be an anti-proliferative agentin taxane (for example docetaxel or cabazitaxel) resistant cells. Thus,identification of therapy resistance biomarkers may optimize selectionof therapies known to be active by circumventing these resistancemechanisms.

Further, as PCa is a multifactorial disease, a biomarker for optimalgaleterone therapy may be in the form of a panel of biomarkers or abiomarker panel that are indicators of disease status, therapeuticoptimization, and outcomes. Galeterone may be prescribed alone or incombination with another therapy, thus a biomarker, biomarkers, or abiomarker panel can help predict utility, efficacy, safety, or toxicityand can help guide optimal medical decision making for not onlygaleterone alone, but galeterone in combination with other therapies.

Biomarkers can be prognostic, predictive, pharmacodynamic/mechanistic,or surrogate. Prognostic biomarkers are predictive of likely outcome ofa disease independent of treatment. In PCa, prognostic biomarkersinclude PSA level, Gleason score, monitoring pattern of spread ofdisease, presence/absence/morphology/enumeration of CTCs, lactatedehydrogenase levels, and pain. Predictive biomarkers come in the formof a disease or host characteristic that may be involved in theimprovement of outcomes with a particular treatment; for PCa predictivebiomarkers include: PSA levels, when to biopsy, when to re-biopsy, whento start treatment and when to alter treatment. Pharmacodynamicbiomarkers are able to reveal the mechanism of action or result of apharmaceutical intervention, and in PCa these may include loss ofandrogen (testosterone or DHT), up or down regulation of AR specificgene expression (i.e. protein analysis), immune reactivity (detection ofantibody or specific immune cells), PSA levels, or broadly, tumorshrinkage. Surrogate biomarkers are used to estimate the treatmenteffect as an intermediate endpoint for a gold standard outcome (eg.survival). In PCa, surrogate markers include CTC enumeration, PSAreduction, radiographic progression free survival. Overall, biomarkerspose a rational approach to addressing current clinical challengesincluding when and in which patient to biopsy or re-biopsy, offerinterventional therapies (surgery and the like), or alter/combinetherapies. Identifying a biomarker or a panel of biomarkers forcompanion use in galeterone therapy will provide powerful decisionmaking capability to the therapeutic management of disease. Whilepredictive and surrogate biomarkers carry a greater degree of importancein therapeutic management and decision making, either or both combinedwith prognostic biomarkers may provide even greater value to clinicalmanagement.

Prognostic factors for post-treatment include PSA decline relative topretreatment levels, pain improvements, quality of life improvements(direct patient measure), changes in CTC count (for example greater than5 to less than 5 per mL blood sample) and changes in CTCcharacterization (for example, size of CTCs), PSA Progression FreeSurvival (PFS), radiographic PFS, induction of immunity to tumorantigens (sipuleucel-T). PCa has a high propensity for bone metastasisand it is postulated that this is mediated through the acquisition ofosteomimicry or adhesion molecules that allow attachment to the bonemicroenvironment. Agents such as zoledronic and denosumab may interferewith tumor bone stromal interactions and thus limit the skeletal relatedevents (SREs) such as fractures, radiation/surgery to the bones, andspinal cord compression. Effects of PCa bone metastases can beindirectly related to bone turnover markers such as the bone type 1collagen breakdown product N-telopeptide (urine/serum Ntx) and otherssuch as tartrate-resistant acid phosphatase 5b, serum type 1 Ctelopeptide, osteopontin. Other biomarkers might include osteoclastactivators such as bone alkaline phosphatase (BAP, or a component oftotal alkaline phosphatase), or broadly one or more of theOPG/RANKIJRANK system, a dominant, final mediator of osteoclastogenesis.Another clinical consequence of PCa is anemia brought on by bone marrowsuppression. While anemia might be a consequence of androgen deprivationtherapy, renal disease, chemotherapy toxicity, anemia of chronicdisease, iron deficiency from blood loss, bone marrow infiltration orother co-existing disease, hemoglobin levels and anemia has been shownto be a prognostic factor in the nomogram CRPC risk-based classification(anemia, progression by bone scan, visceral metastases, pain). Anemiafalls into the category of reflecting both burden of PCa as well as hostresponse.

Another leading prognostic indicator of PCa is lactate dehydrogenase(LDH). Elevations of LDH are thought to be reflective of the underlyingtumor burden or of an aggressive tumor phenotype. It is thought that LDHlevels are useful for the clinical stratification of randomized patientsin clinical trials and use for prognostic decision making.

Androgen receptor activity and gene expression profiling has beenstudied in prostate cancer. In seeking a biomarker, one begins withidentifying an up-regulated gene and testing if this gene product can bea candidate biomarker. Gene expression profiling and linking theexpression to mechanism of therapeutic resistance has been described byHolzbeierlein et al, Am. J. Path. 164(1), pp 217-227 2004. Whileenhanced or reduced expression of certain genes have been identified,genomic alterations in certain genes may also occur in prostate cancerand these include: rearrangement (ETS transcription factors, RAF, KRAS);mutation (androgen receptor, PIK3CA, AKT, RAF, KRAS); amplification(androgen receptor, PIK3CA, MYC, AURKA); loss (PTEN, RB 1). Other knowngenetic alterations occur in the SPOP, FOXA 1, AURKA, MED 12, MAGI-1 andCHD 1 genes. ETS fusions can be found it upwards of 50% of PCa and atargeted therapy or biomarker may be useful, for example targetinginhibition of PARP or DNAPK or analyzing patient samples for the ETSfusions. Oncogene expression, RAS/RAF, MYC, as well as the tumorsuppressor gene RB 1 may be useful biomarkers.

Androgen receptor is known to regulate a large repertoire of genescentral to the identity and behavior of prostate cancer cells.Overexpression of long non-coding RNA, for example PCGEM 1 and PRNCR1,is associated and has been correlated with susceptibility of prostatecancer. Recently it was reported that both PCGEM 1 and PRNCR1 are highlyoverexpressed in CRPC and they bind to and activate both liganddependent and ligand independent AR-mediated gene activation programsand can lead to unchecked proliferation in prostate cancer cells. (Yanget al. Nature 2013, 500(7464):598-602)

Prostate cancer biology varies from locally confined tumors with lowrisk for relapse to tumors with high risk for progression. Currently,few biomarkers are in use for patients with prostate cancer. Forexample, Gleason score and serum prostate specific antigen (PSA) levelsare used separately to predict pathological stage in patients withlocalized prostate cancer. Because the degree of tumor differentiationhas a profound influence on the expression of serum PSA, serum PSAlevels alone do not reflect tumor burden accurately. CTC may have thepotential to accurately and independently predict aspects of PCa andstudies have linked identifying CTC to overall survival in CRPC. Otherstudies have pointed to the development of a panel of biomarkers such asa correlation of expression patterns of some biologically relevantproteins with clinically relevant scoring for example androgen receptor(AR) co-activators, lysine-specific histone demethylase 1 (LSD 1) andfour and a half LIM-domain protein 2 (FHL2), AR, p53 along with Gleasonscore, Gleason grade and PSA levels.

Biomarkers for CRPC, a stage of PCa for which few therapeutic optionsexist, include: development of visceral metastatic disease (e.g. kidney,brain, intestine, pancreas, colon, lung, adrenal, breast, livermetastases), performance status, pain, hemoglobin, anemia, alkalinephosphatase (bone), pain, PSA and PSA by RT-PCR, PSA kinetics, CTCcount, lactate dehydrogenase, albumin, type of progression (i.e. bone,measurable disease, or PSA elevation only), VEGF levels, IL-6 levels,chromagranin-A levels, serum TRAP-5b and other bone turnover markers(sCTX, P1NP, and others), Gleason sum in primary tumor, and urineN-telopeptide.

A biomarker or a biomarker panel is a measured characteristic,substance, or analyte or group of characteristics, substances oranalytes that are objectively measured and evaluated as an indicator ofnormal biological processes, pathogenic processes, or pharmacologicresponses to a therapeutic intervention. Cancer staging, includingidentification and/or localization of tumor, nodes, and or metastases(TNM), may the broadest clinical set of biomarkers. Biomarkers can bereadily attained from patient samples for routine monitoring and thusbiomarkers analyzed in whole blood, serum or plasma, urine, mucous,feces, tears, semen and the like are most easily obtainable. However, insome cases, biomarkers may be analyzed in patient samples that requiremore invasive procedures such as biopsy or tissue sampling for exampletumor, bone, skin, teeth, organ biopsy (liver, kidney, colon, lungpancreas). Alternatively, circulating tumor cells or exosomes fromprostate tumor or metastatic cells may be tested for biomarkers.Biomarkers include biological, physiological molecules, compounds,substances, or analytes and are analyzed to determine anabsence/presence, level, concentration, value, intensity, activity, ormeasurement.

Employing the least invasive procedure to analyze a biomarker is mostfavorable, such as imaging methods and specific detection or evaluationemploying imaging analyses may be employed, such as biomarker specificnanoparticles or magnetic nanoparticles, radioactive substances, orother tools to specifically image a biomarker in vivo. Biomarkers may bedetected using standard methods known in the art, including:immunodetection, PCR (realtime PCR, RT-PCR, qPCR, TaqMan PCR),chromatography, mass spectrometry, NMR and the like. Biomarkers stemmingfrom gene expression assays using RNA isolated or derived from a patientor subject sample, may include RNA quantification, RNA QC and reversetranscription, DNase 1 treatment and PCR based quantitative geneexpression analysis and microRNA assays. The gene expression analysismay include high, medium or low density arrays or a combination of geneexpression arrays, and these analyses are focused on allowing foranalysis of gene expression pattern identification across many genesthat are known to be induced by androgen receptor activation. Biomarkersmay be characteristic of a pathogenic processes and may include ameasurement of health quality of life, such as pain, ease and frequencyof urination, sexual function, and the like.

Androgen Receptor and Androgen Receptor Variants as Biomarkers

The androgen receptor gene has a cytogenetic location at Xq12 and themolecular location on the X chromosome is at base pairs 67,544,031 to67/730,618. Androgen receptor is currently understood to consist of 8exons (see FIG. 1). Exon 1 encodes amino-terminal domain (NTD)containing transcriptional activation sites; exons 2-4 encodeDNA-binding domain (DBD); while exons 5-8 encode a Ligand-binding domain(LBD). Alternative spliced variants exist, for example, lacking the LBD.Such variants may be constitutively active. A splice variant lacking theLBD may, for example, localize in the nucleus, where it binds DNA andactivates transcription independently of ligands.

The androgen receptor gene contains two polymorphic trinucleotidemicrosatellites in exon 1. The first microsatellite (nearest the 5′ end)contains 8 to 60 repetitions of the glutamine codon “CAG” and is thusknown as the polyglutamine tract. The second microsatellite contains 4to 31 repetitions of the glycine codon “GGC” and is known as thepolyglycine tract. In the polyglutamine tract normally, the number ofCAG repeats in the AR gene ranges from fewer than 10 to about 36 and inCaucasian men the average number is 21 and in Black men the averagenumber is 18. In prostate cancer, some studies have shown an increasedrisk of prostate cancer in men with a short CAG repeat, and extra copiesof the gene in cancer cells may be associated with progression of thedisease. Spinal and bulbar atrophy (Kennedy's disease) results from anexpansion of the CAG trinucleotide repeat in the AR gene. In Kennedy'sdisease, CAGs are abnormally repeated from 38 to more than 60 times. Inbreast cancer it has been suggested that a long CAG repeat region isassociated with an increased risk of breast cancer in women, and that ashorter CAG repeat region is associated with a reduced risk. Otherresearch indicates that a shorter CAG repeat region may be related to anincreased risk of both breast cancer and benign breast disease. ShorterCAG repeat regions have also been associated with more aggressive formsof breast cancer. Further, a longer CAG repeat region in the AR gene mayincrease the risk of endometrial cancer in women. Although the extendedCAG region changes the structure of the androgen receptor, it is unclearhow the altered protein disrupts cells and androgen-AR mediatedintracellular response. A fragment of the androgen receptor proteincontaining the CAG repeats appears to accumulate within cells and theaccumulation may interfere with normal cell functions. This buildup maylead to apoptosis and in Kennedy's disease there is nerve cell loss inthe brain and spinal cord that control muscle movement. In contrast, aprostate cancer cell, or a cell having low CAG repeats in the AR geneproduct, has circumvented the buildup of AR protein fragments by havinglow number of CAG repeats in the AR gene and hence a more likely processof AR protein expression and functionality. Thus, identification of thenumber of CAG repeats in the AR gene product may provide a formidablebiomarker of androgen-dependent disease or therapy of androgen-dependentdisease.

Prostate cancer is an androgen receptor dependent disease. Treatments,as described above, are often aimed at the androgen receptor, ligandbinding to the receptor, or androgen mediated intracellular signalingpathways. PCa has been shown to circumvent these treatment pathways(resistance, resistance to treatment, treatment failure) by processes ofselectivity and treatment pressures, to mutate the key proteins involvedin the proliferation and “health” of the tumor. Mutations or geneticalterations may result in gain or loss of function, increased ordecreased ligand binding, increased or decreased gene expression(changes or selectivity on gene expression of the androgen receptoritself or a gene product that is involved in steroid receptor activity),increase or decrease of steroid receptor DNA binding, receptorconstitutive activity or loss of ligand responsivity, changes to theability of the receptor to dimerize, changes to ligand binding sites onthe effector proteins—e.g. cofactors become enhancers or inhibitors,antagonists become agonists, ligand promiscuity (e.g. progesterone,hydroxycortisone, estrogen, and cortisol under normal conditions do notbind the androgen receptor and ligand binding mutations within theandrogen receptor may allow binding and activation by these otherphysiological relevant steroids). Thus, “altered” or “mutated” or“mutant” androgen receptor is used to refer to an androgen receptorwhich has changed relative to its wild-type form. Fo example, thealtered androgen receptor phenotypically expresses one or more of theabove described gain or loss of function. Changes include, but are notelimited to splice variants including exon skipping, cryptic splicingdonor/acceptor usage, and cryptic exon inclusion; amino acidsubstitution/s, deletion/s, or insertion/s; alterations of posttranscription and/or post translation processing (i.e. glycosylation,folding, phosphorylation, ubiquinylation or the like).

Biomarkers for prostate cancer include the expression variants of theandrogen receptor and known mutations have been found in establishedcell lines or from tumor biopsies. Mutations can be amino acidsubstitutions, insertions or deletions. Alternatively, there are splicevariants that have been identified. Exemplary mutations include, forexample, E43G, L54S, Q58L, L57Q, Q64R, AQ86, Q112H, G142V, E166S, K180R,L192F, Q198G, E211E, D221H, N222D, T227C, M266T, P269S, A251V, E253K,S296R, P334F, P340L, A356V, P390L, G414S, W433L, T438P, T438I, L444S,G449D, G451D, G456S, G457D, R484C, T497I, A498T, P499P, V508L, G524S,G524D, D528G, AL547, AP554, T573A, L574P, K580R, A586V, A587S, L594M,K609E, R629Q, K630T, S646D, S647N, E665D, Q670R, I672T, G683A, V716M,V715M, L701H, L720E, A721T, V730M, R726L, L744V, A748V, M749I, G750S,F754L, T755A, V757A, S759P, Y763C, W741C, F747L, N756A, V757I, R760K,W741X, AG743, W751X, S782N, R786X, W7960, L797P, Q798E, S791P, I799P,L830P, R846G, Q867X, H874Y, T877A, T877S, V866M, L880Q, L872P, D879G,M886I, A896T, Q902R, F891L, G909Q, Q919R, D890N, M895V, and K910R. Forexample, the amino acid substitutions are: T877A (T878A), D879G (D878G),W741C, W741L, M749L, R629Q, G142V, P533S, T575A, H874Y or, F876L. Thesepoint mutations may be categorized into the three main regions of thesteroid receptor protein 1) LBD mutants (T877A, D879G, W741C. W741L,M749L, H874Y, F876L) and mutations in the LBD may have altered ligandbinding due to receptor protein conformation changes or alterations inamino acid R groups in the ligand binding pocket or conformationresulting in loss of ligand binding, loss of ligand recognition,switching of antagonist to agonist, and/or ligand promiscuity; 2) NTD orhinge region mutants (R629Q, G142V, P533S) that may affect the abilityof receptor transactivation, interaction with the transcriptionmachinery or cofactors/regulators and result in alterations of receptorfunctions such as DNA binding, regulating gene expression, or nucleartranslocation; or 3) DBD mutants (T575A) that may affect the receptor'sability to regulate of gene expression. Examples include: H874Y mutationin the androgen receptor has been shown to allow estradiol,progesterone, hydrocortisone, flutamide, and bicalutamide binding in22Rv1 and CWR22RV1 cells; D878G has been shown to confer loss of DHT andtestosterone binding and activity; W741C mutations confers bicalutamideand flutamide as agonists; F876L changes ARN-509 and enzalutamide fromantagonists to agonists; M749L confers a hypersensitivity to estradiol;T575A leads to preferential binding to AR-nonspecific motifs, i.e. GRE;R629Q leads to gain of function with DHT.

Splice variants include exon skipping, cryptic splicing donor/acceptorusage, and cryptic exon inclusion. Variants that have been identifiedinclude AR-V1, AR-V2, AR-V3, AR-V4. AR-V5, AR-V6, ARV7, AR-V567es,AR-V7, AR-V9, AR-V12, AR-V13, and AR-V14. See, e.g. US PatentApplication No. 2011/0110926, U.S. Pat. No. 8,133,724, and US PatentApplication No. 2013/0130241). Generally, the androgen receptor variantsare lacking some or all of the LBD and/or that portion of the carboxylterminal of the androgen receptor protein that confers ligand binding.

In a clinical study of castrate resistant prostate cancer (AntonarakisE, Lu C, Wang H, et al. Androgen Receptor Splice Variant-7 PredictsResistance to Enzalutamide in Patients with Castration ResistantProstate Cancer. 2014 AACR Annual Meeting. Abstract 2910. Presented Apr.7, 2014), 39% of patients expressed AR-V7 mRNA in circulating tumorcells. This subset of patients had a worse prognosis and worse responseto anti-androgen treatment. Specifically, these patients showed no PSAdecline when treated with enzalutamide and had shorter time toprogression (2.1 months) relative to AR-V7 negative patients, for whichPSA levels dropped by 50% in 53% of patients, and which had a longertime to progression (6.1 months). In some embodiments, the presence ofthe androgen AR-V7 variant correlates with resistance to anti-androgenssuch as enzalutamide (Efstathiou et al. European Urology 2014). In someembodiments, galeterone is administered to subjects identified to havetumors in which AR-V7 is expressed. In other embodiments, galeterone isadministered to subjects identified to have tumors in which an androgenreceptor is expressed which has a carboxy terminal loss.

Another variant known as AR-V12, has been shown to have about a 40%prevalence in androgen receptor positive metastatic samples. Since ithas been proposed that nearly 60% of androgen receptor positivemetastasis samples express one or more androgen receptor splicevariants, it would follow that a large percentage of the tumors wouldthen have the AR-V12 splice variant. Further, AR-V12 has been observedin samples from men that have demonstrated resistance to abiraterone(Mostaghel et al. 2011). The AR-V12 variant has been shown to beconstitutively active, as it is lacking the ligand binding domain.

Another such variant is the AR point mutation T878A (alternatively“T877A”), which is reported in 33% of hormone-refractory tumors. Thismutation increases the promiscuity of AR, allowing progesterone, whichis elevated in patients treated with abiraterone, to activate AR-T878A.The tumor therefore continues to grow despite continued androgenblockade and abiraterone resistance can be conferred by expression ofthis mutant AR variant. The mutation also changes the bindingspecificity of the receptor such that in tumors carrying this mutation,flutamide acts as an agonist, while bicalutamide loses its activity.AR-T878A has a 6-fold increase in activity relative to wild type AR. Insome embodiments, the presence of the androgen AR-T878A variantcorrelates with resistance to anti-androgens such as enzalutamide andabiraterone. In some embodiments, galeterone is administered to subjectsidentified to have tumors in which AR-T878A is expressed.

Another such variant is the AR point mutation F876L. This single aminoacid mutation is within the AR LBD and it has shown to affect bothenzalutamide and ARN-509 binding and ultimately potentially mediatestumor resistance to both compounds. The F876L mutation was identified inapproximately 10% of ARN-509 treated patients. It has been postulatedthat the F876L mutation switches an antagonist to agonist effects andhence the mutation drives resistance to the anti-androgen compound.

In some embodiments, an AR variant is detected in a tumor sampleisolated from a subject. For example, circulating tumor blood cells areisolated from the subject the cells are tested for the presence of theAR variant. In one embodiment, cells are tested for immunoreactivity ofan antibody to bind the C-terminal portion (e.g. the ligand-bindingdomain) of the AR protein. For example, an antibody having specificityto the ligand-binding domain is used. Lack of immunoreactivity indicatesthe presence of an AR variant which lacks the ligand-binding domain,while presence of immunoreactivity indicates presence of an AR proteinwhich possesses the ligand-binding domain, including the wild-type ARprotein. The analysis can also be performed using an antibody specificto the NH2 portion of the AR protein, thus it can detect the presence ofall AR proteins, whether they have a C-terminal truncation or not. Inanother embodiments, detection of the AR variant is performed bydetecting the presence or level of a nucleic acid (including DNA or RNA)coding for the AR variant. Detection is performed, for example, by usinga nucleic acid amplification reaction, a gene expression assay, or byusing a sequencing-based method. In some embodiments, detection of ARV-7is performed as described in US Patent Application NO. 2011/0110926,filed on Jan. 18, 2011, which is hereby incorporated by reference in itsentirety. For example, PCR amplification of an RNA transcript isperformed using primers designed to amplify the truncated ARV-7transcript. In some embodiments, quantitative PCR amplification isperformed.

Resistance to taxanes in PCa has been observed to be correlated withantiandrogen insensitivity. Further, taxanes have been shown to have adifferential effect on prostate cancer cells that are expressing ARv567vs ARv7, which are clinically relevant splice variants. ARv567,appearing in about 59% of prostate cancer tumor specimens from patientshaving CRPC and arises in response to ADT or abiraterone therapy,appears to have an effect on dynamic microtubules and the dynein motorprotein. Hence the AR variant ARv567 is sequestered in the cytoplasm andthus is inactive in promoting transcription in the presence of taxanetherapy. In contrast, ARv7 is present in both benign and malignantprostate tissues, but has been described as enriched in metastaticdisease. In a recent study, ARv7, a variant that lacks the hinge region,did not co-sediment with microtubules or co-precipitate with dyneinmotor protein and both nuclear accumulation and transcriptional activityof ARv7 was unaffected by the presence of taxanes. (Thadani-Mulero etal. Cancer Res. 74(8):2270-2282) In some embodiments, galeterone isadministered to subjects identified to have tumors resistant to taxanesby analyzing a biomarker in the subject for one or more AR mutants.

Abiraterone, a CYP17 inhibitor, reduces CRPC growth via suppression ofintratumoral androgens. Resistance to abiraterone may occur throughupregulation of CYP17A1 and/or induction of androgen receptor and ARsplice variants that confer ligand independent signaling. In someembodiments, galeterone is administered to subjects identified to havetumors resistant to abiraterone.

Epithelial-mesenchymal transitions (EMTs) occur as key steps duringembryonic morphogenesis, and are now implicated in the progression ofprimary tumors towards metastases. EMTs in prostate cancer are areasonable candidate for progression to CRPC. Recent advances havefostered a more detailed understanding of molecular mechanisms andnetworks governing EMT in tumor progression. Besides TGFβ and RTK/Rassignaling, autocrine factors and Wnt-, Notch-, Hedgehog- andNF-κB-dependent pathways were found to contribute to EMT. Repression ofE-cadherin by transcriptional regulators such as Snail or Twist emergesas one critical step driving EMT, and this stage is currently beingmolecularly linked with many of the new players. Increasing evidencesuggests that EMT plays a specific role in the migration of cells from aprimary tumor into the circulation and may provide a rationale fordeveloping more effective cancer therapies or for understandingmetastasis.

One of the main limitations in evaluating treatments for metastatic PCais the inability to use available clinical imaging modalities to assesstreatment response in bone, which is the predominant and often the onlysite of metastasis in 85% to 90% of patients. Traditional clinicalassessment of bony metastases is achieved through radionuclide bonescintigraphy. Although the use of bone scintigraphy (bone scan),ultrasound, positron emission tomography (PET),(18)F-16beta-fluoro-5alpha-dihydrotestosterone ((18)F-FDHT) PET inprostate cancer patients undergoing therapy, computed tomography (CT),endorectal coil magnetic resonance imaging, and magnetic resonanceimaging (MRI) plays a distinct role in identifying and characterizingthe extent of disease. The use of diffusion MRI for assessing responseto anticancer therapy is based on its ability to quantify the random orBrownian motion of water. Diffusion of water within a tumor is reducedin the presence of cellular membranes that act to impede the randommotion of water molecules. During the course of successful treatment,loss of tumor cells and/or tumor cell membrane integrity occurs, whichwill then result in a reduction in the barriers that impede mobility ofwater molecules. Diffusion MRI can be used to assess the treatmenteffect through quantification of the amount of increased apparentdiffusion coefficient (ADC) values in tumor regions experiencing a lossof cellular density. Thus, water mobility within a tumor will increaseover time following effective treatment, as represented by an increasein MRI-quantified ADC values, with the magnitude of the change relatedto the effectiveness of the therapy. An alternative post-processingapproach known as the functional diffusion map (fDM) was developed tostandardize the processing of clinical diffusion MRI data to provide fora sensitive and quantifiable means for early assessment of cancertreatment outcome. The fDM approach of monitoring anticancer therapyallows spatial, voxel by voxel tracking of changes in tumor waterdiffusion values over time. Changes in diffusion values are depicted infDM images by color encoding of tumor diffusion voxels that were altereddue to therapy (either increased or decreased ADC value), therebyallowing for a spatially resolved analysis of ADC within an individuallesion.

By sampling blood and tumor biopsies, or using imaging techniques, it ispossible to identify specific markers, e.g. one or more biologicallyrelevant species that when analyzed have the potential to predictwhether the patient will respond to galeterone. These biomarkerscomprise molecular and cellular markers and include:

-   -   a. Genomic sequencing of specific genes within tumor cells or        CTCs from a patient (ex. TP53, ZFHX3, RP1, PTEN, TMPRSS2 fusion,        APC, wnt signaling, AR mutations and truncations, AR        amplification, hepsin, PIM-1)    -   b. PTEN is a tumor suppressor gene that is involved in cell        cycle regulation and is consistently associated with poor        prognosis in PCa. Deletion of PTEN is associated with a higher        Gleason grade, risk of progression, advanced localized or        metastatic disease, and recurrence after therapy. Typically        measured by fluorescence in situ hybridization (FISH), the test        is typically ordered in conjunction with biopsy tests to        indicate partial (hemizygous) or complete (homozygous) deletions        of the gene    -   c. Mutations in the androgen receptor:        -   i. Point mutations leading to amino acid substitutions,            including: T877A (T878A), D879G (D878G), W741C, W741L,            M749L, R629Q, G142V, P533S, T575A, H874Y, F876L        -   ii. Splice variants, including AR-V1, AR-V2, AR-V3, AR-V4.            AR-V5, AR-V567es, AR-V6, AR-V7, ARV7, AR-V9, AR-V12, AR-V13,            AR-V14. (see patents US2011/0110926, U.S. Pat. No.            8,133,724, US2013/0130241).    -   d. Circulating tumor cells (CTC)    -   e. Enumeration at baseline (circulating cells with the profile:        CK+, CD45−) or at some time after initiation of therapy    -   f. Enumeration of a sub-set of CTC which are small in size or by        another cellular shape/size characteristic at baseline or at        some time after initiation of therapy    -   g. Enumeration of CK−, CD45− CTC candidates at baseline or at        some time after initiation of therapy    -   h. Androgen receptor (AR) expression in CTC or tumor biopsies at        baseline or at some time after initiation of therapy    -   i. Sub-cellular localization of AR protein in CTC or tumor        biopsies (nuclear: cytoplasmic ratio) at baseline or at some        time after initiation of therapy    -   j. Assessing deletions and gene rearrangements in CTC and tumor        biopsies by FISH (ex. PTEN deletion, TMPRSS2 fusions) at        baseline or at some time after initiation of therapy    -   k. TMPRSS2-ERG is a fusion between the transmembrane protease        serine 2 gene and the v-ets erythroblastosis virus E26 oncogene        homolog (avian (ERG) gene. This gene fusion is the predominant        variant in approximately 40-80% of PCa. Quantitative levels of        TMPRSS2-ERG in the urine appears to be associated with        clinically significant PCa based on the Epstein criteria—a        stratification of disease aggressiveness using PSA density and        characteristics of the patient's biopsy (Gleason score), the %        of tumor vs normal tissue observed, and number of cores with the        tumor. TMPRRSS2-ERG detection combined with detection of PCA3 in        urine has shown to have utility in predicting the severity of        the PCa.    -   l. Absolute Prostate specific antigen (PSA) blood level and PSA        doubling time prior to treatment    -   m. Prostate specific membrane antigen (PSMA) expression as        determined by imaging modalities such as radiolabeled ligands of        PSMA or antibodies than bind PSMA.    -   n. 5 Kallikrein panel (total PSA, free PSA, intact PSA,        Kallikrein 2)    -   o. Pre-treatment testosterone blood level    -   p. Changes in steroid levels at some time after initiation of        therapy. Steroids include: androgens (testosterone, DHT),        androgen precursors (DHEA, DHEA-Sulfate, androstenedione),        corticosteroids, progestogens, mineralocorticoids, and androgen        precursors in the “back-door” pathway.    -   q. Staging of prostate tumor via Gleason Score (a biopsy grading        scale from 1-5; lower Gleason grades are associated with small,        closely packed glands and cells spread out and lose glandular        architecture as Gleason grade increases)    -   r. Metabolic markers such as P450 enzymes that may be used to        determine hi-, med-, low-metabolizers    -   s. CYP17 mutations that may change the efficacy of galeterone    -   t. Immune checkpoint blockade and immunologic approaches (CTLA-4        blockade)    -   u. Immune modulators, programmed death ligands 1 and 2 (PD-L1        and PD-L2) and their receptor, for example PD-1.    -   v. Prostate health index—a ratio of pro-PSA to free PSA    -   w. PCA3—a noncoding mRNA that has been shown to be elevated        in >90% of men with PCa. PCA3 is measured in urine    -   x. Prostate core mitomic test—identifies a large-scale depletion        in mitochondrial DNA that indicates cellular change associated        with undiagnosed prostate cancer and detects the presence of        malignant cells in normal appearing prostate tissue across an        extended area.    -   y. CCP genes—cell cycle progression gene mutations    -   z. Serologic tests include: hemoglobin, lactate dehydrogenase        (LDH), alkaline phosphatase.    -   aa. Cross reactivity in resistance—chemotherapy resistance and        enzalutamide resistance appears to be common in CRPC therefore a        biomarker that suggests chemotherapy resistance may also        indicate a resistance to enzalutamide or to the broader class of        anti-androgen compounds.

By sampling blood and tumor biopsies, or using imaging techniques, itmay be possible to determine whether a patient is responding togaleterone therapy. Markers that are measured include:

-   -   a. Decrease in numbers of CTC (particularly after 1 week of        galeterone therapy)    -   b. Increase in apoptotic CTC    -   c. Decrease in PSA or reduction in PSA doubling time    -   d. Increase in PSMA expression as determined by imaging        modalities such as radiolabeled ligands of PSMA or antibodies        than bind PSMA. Because blocking androgen-signaling results in        an increase PSMA expression, an increase in the PSMA signal is        an indicator of anti-androgen activity.    -   e. Reduction in the tumor ¹⁸F-DHT-PET signal, indicating        antagonism of the androgen receptor    -   f. ProMark—a tissue biopsy based test—differentiation of        indolent from aggressive disease in formalin-fixed,        paraffin-embedded tissue samples.    -   g. Proteosome degradation pathway members—i.e. inhibition of the        tagging or removal of the androgen receptor from the cell

Described herein, in certain embodiments, are compounds, methods ofmaking such compounds, pharmaceutical compositions and medicamentscomprising such compounds, and methods of using such compounds to treatandrogen receptor mediated diseases or conditions including, but notlimited to, prostate cancer and benign prostatic hyperplasia. In someembodiments, the androgen receptor mediated disease or condition isprostate cancer. In some embodiments, the prostate cancer is castrationresistant prostate cancer.

In some embodiments, the disease is an anti-androgen resistant disease.For example, the anti-androgen resistant disease may have previouslybeen treated by providing an anti-androgen therapy, such as, e.g.,castration, treatment with an androgen receptor antagonist, or acombination thereof. The disease may have initially responded to theanti-androgen therapy, but subsequently become insensitive to thetherapy (e.g., worsened despite continued anti-androgen treatment). Insome embodiments, the disease may have always been insensitive to theanti-androgen therapy.

In some embodiments, the disease is an androgen dependent disease and ismarked by excessive production of adrenal or gonadal androgens byadrenal adenomas. carcinomas, or hyperplasia, Leydig cell tumors in men,and arrhenoblastomas and polycystic ovarian syndrome in women. Androgendependent disease includes Kennedy's disease, breast cancer, prostatecancer, bladder cancer, pancreatic cancer, ovarian cancer, acne,hidradennitis supprurativa, androgenic alopecia, keratosis pilaris,begin prostatic hyperplasia, hisutism.

In some embodiments, the invention provides compounds, pharmaceuticalcompositions, and medicaments comprising such compounds, and methods ofusing such compounds that decrease androgen biosynthesis, decreaseandrogen receptor signaling and decrease androgen receptor sensitivity.

Also contemplated is a method of treating a disease by administering toa subject in need thereof a combination therapy comprising ananti-androgen therapy and a compound of Formula I, II, and/or III. Theanti-androgen therapy can be, e.g., castration, treatment with anandrogen receptor antagonist, e.g., enzalutamide, ARN-509, vinclozolin,procymidone, linuron, the DDT metabolitedichlorodiphenyldichloroethylene (p.p′-DDE), ketoconazole, fenitrothion,Di-n-butyl phthalate (DBP), diisobutyl phthalate (DiBP), benzyl butylphthalate (BBP), Bis(2-ethylhexyl) phthalate (DEHP) and di-n-pentylphthalate (DPP), Paraben esters, such as butylparaben,3,3′-Diindolylmethane (DIM), Scutellaria baicalensis,N-butylbenzene-sulfonamide (NBBS), atraric acid, bicalutamide,flutamide, spironolactone, cyproterone acetate, finasteride,dutasteride, and nilutamide, docetaxel, cabazitaxel, a taxane, or anycombination thereof.

In some embodiments, the compound of Formula I or formula II and theanti-androgen therapy are administered sequentially, simultaneously,alone, or in combination. In some embodiments, the compound of Formula Iand the anti-androgen therapy (e.g., the androgen receptor antagonist)are formulated into the same pharmaceutical composition foradministration to the subject.

In one aspect, the compounds, pharmaceutical compositions andmedicaments comprising such compounds, and methods of using suchcompounds decrease androgen biosynthesis. In some embodiments, thecompounds disclosed herein inhibit the activity of enzymes that controlsandrogen production. In certain embodiments, the compounds disclosedherein inhibit the activity of cytochromeC_(17α)-hydroxylase/C₁₇₋₂₀-lyase (CYP17).

In one aspect, the compounds, pharmaceutical compositions andmedicaments comprising such compounds, and methods of using suchcompounds decrease androgen receptor signaling. In some embodiments, thecompounds disclosed herein bind to the AR and are a competitiveinhibitor of testosterone binding.

In one aspect, the compounds, pharmaceutical compositions andmedicaments comprising such compounds, and methods of using suchcompounds decrease androgen receptor sensitivity. In some embodiments,the compounds disclosed herein reduce the content of AR protein withinthe cell and diminish the ability of the cell to be sustained by lowlevels of androgenic growth signals.

Compounds

In one aspect, the invention provides compositions comprising a compoundof Formula I

or a pharmaceutically acceptable salt, N-oxide, active metabolite,prodrug, or solvate thereof; wherein R₁ is H or acetyl; R₂ is pyridyl orbenzimidazole.

In some embodiments, the compound is a compound of Formula II (alsoknown as “galeterone”; “TOK-001”; or “VN/124-1”):

or a pharmaceutically acceptable salt, N-oxide, active metabolite,prodrug, or solvate thereof.

In other embodiments, the compound is a compound of Formula III:

-   or a pharmaceutically acceptable salt, N-oxide, active metabolite,    prodrug, or solvate thereof;

The compounds of Formula I-III, pharmaceutically acceptable salts,pharmaceutically acceptable N-oxides, pharmaceutically activemetabolites, pharmaceutically acceptable prodrugs, pharmaceuticallyacceptable polymorphs and pharmaceutically acceptable solvates thereof,modulate the activity of steroid hormone nuclear receptors and, as such,are useful for treating androgen receptor mediated diseases orconditions.

Exemplary Synthesis of the Compounds

Compounds of Formula (II) (also described as Compound (1) or3-β-Hydroxyl 17-(1H-benzimidazol-1-yl)androsta-5,16-diene) or TOK-001 orGaleterone) may be synthesized using standard synthetic techniques knownto those of skill in the art or using methods known in the art incombination with methods described herein. Compounds of Formula (III)may be synthesized by similar methods. As one of skill in the art wouldunderstand, the solvents, temperatures and reaction conditions presentedherein may vary according to the practice and knowledge of those ofskill in the art.

The starting material used for the synthesis of the Compound (1) can beobtained from commercial sources, such as Aldrich Chemical Co.(Milwaukee, Wis.), Sigma Chemical Co. (St. Louis, Mo.), or the startingmaterials can be synthesized. The compounds described herein, and otherrelated compounds having different substituents can be synthesized usingtechniques and materials known to those of skill in the art, such asdescribed, for example, in March, ADVANCED ORGANIC CHEMISTRY 4^(th) Ed.,(Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY 4^(th) Ed.,Vols. A and B (Plenum 2000, 2001), and Green and Wuts, PROTECTIVE GROUPSIN ORGANIC SYNTHESIS 3^(rd) Ed., (Wiley 1999) (all of which areincorporated by reference in their entirety). General methods for thepreparation of compounds as disclosed herein may be derived from knownreactions in the field, and the reactions may be modified by the use ofappropriate reagents and conditions, as would be recognized by theskilled person, for the introduction of the various moieties found inthe formulae as provided herein.

Compounds of Formula I-III can be prepared as a pharmaceuticallyacceptable acid addition salt (which is a type of a pharmaceuticallyacceptable salt) by reacting the free base form of the compound with apharmaceutically acceptable inorganic or organic acid, including, butnot limited to, inorganic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, metaphosphoric acid,and the like; and organic acids such as acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, p-toluenesulfonic acid, tartaric acid, trifluoroaceticacid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid,cinnamic acid, mandelic acid, arylsulfonic acid, methanesulfonic acid,ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonicacid, benzenesulfonic acid, 2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, and muconic acid.

Compounds of Formula I-III can be prepared as a prodrug. Prodrugs aregenerally drug precursors that, following administration to a subjectand subsequent absorption, are converted to an active, or a more activespecies via some process, such as conversion by a metabolic pathway.Some prodrugs have a chemical group present on the prodrug that rendersit less active and/or confers solubility or some other property to thedrug. Once the chemical group has been cleaved and/or modified from theprodrug the active drug is generated. Prodrugs are often useful because,in some situations, they may be easier to administer than the parentdrug. Prodrugs may, for instance, be bioavailable by oral administrationwhereas the parent is not. The prodrug may also have improved solubilityin pharmaceutical compositions over the parent drug. An example, withoutlimitation, of a prodrug would be a derivative of Formula (I-III), whichis administered as a hydrophilic ester (the “prodrug”) to facilitateabsorption in the gastrointestinal tract where improved water solubilityis beneficial, but which then is metabolically hydrolyzed to acarboxylic acid and the active entity, Formula (I-III). A furtherexample of a prodrug is a short peptide bonded to the hydroxyl group ofCompound (1), wherein the peptide is metabolized to provide a compoundof Formula I, II, or III.

Prodrugs may be designed as reversible drug derivatives for use asmodifiers to enhance drug transport to site-specific tissues. The designof prodrugs to date has been to increase the effective water solubilityof the therapeutic compound for targeting to regions where water is theprincipal solvent. See, e.g., Fedorak et al., Am. J Physiol.,269:G210-218 (1995); McLoed et al., Gastroenterol, 106:405-413 (1994);Hochhaus et al., Biomed. Chrom., 6:283-286 (1992); J. Larsen and H.Bundgaard, Int. J. Pharmaceutics, 37, 87 (1987); J. Larsen et al., Int.J Pharmaceutics, 47, 103 (1988); Sinkula et al., J. Pharm. Sci.,64:181-210 (1975); T. Higuchi and V. Stella, Pro-drugs as Novel DeliverySystems, Vol. 14 of the A.C.S. Symposium Series; and Edward B. Roche,Bioreversible Carriers in Drug Design, American PharmaceuticalAssociation and Pergamon Press, 1987, all incorporated herein in theirentirety.

Additionally, prodrug derivatives of compounds of Formula I-III can beprepared by methods known to those of ordinary skill in the art (e.g.,for further details see Saulnier et al., (1994), Bioorganic andMedicinal Chemistry Letters, Vol. 4, p. 1985). Prodrug forms of theherein described compounds, wherein the prodrug is metabolized in vivoto produce a derivative as set forth herein are included within thescope of the claims. Indeed, some of the herein-described compounds maybe a prodrug for another derivative or active compound.

Sites on the aromatic ring portion of compounds of Formula I-III can besusceptible to various metabolic reactions, therefore incorporation ofappropriate substituents on the aromatic ring structures, for example,halogens, can reduce, minimize or eliminate this metabolic pathway.

Various methods of making compounds of Formula I-III are contemplatedand the following descriptions are provided as non-limiting examples. Insome embodiments, one or more of the following chemical reactions isperformed in an inert atmosphere, for example, nitrogen or argon. Insome embodiments, the temperature of the reaction is monitored. In someembodiments, the reaction is monitored by HPLC or TLC. In someembodiments, the pH of the reaction is monitored. In some embodiments,the temperature of the reaction is controlled. In some embodiments, thepurity of the product is determined by HPLC. In some embodiments, theexperiments are run on small scale, medium scale, large scale,analytical scale, or manufacturing scale. In some embodiments, theproduct is clarified by filtration through a pad comprising one or moreof silica gel and celite.

In some embodiments, the synthesis is performed on large scale. In someembodiments, large scale comprises a scale of about 1 to about 10 kg. Insome embodiments, the synthesis is performed on manufacturing scale. Insome embodiments, manufacturing scale comprises a scale of greater thanabout 10 kg. In some embodiments, manufacturing scale comprises a scaleof about 10 to about 1,000 kg. In some embodiments, manufacturing scalecomprises a scale of about 10 to about 100 kg. In some embodiments,manufacturing scale comprises a scale of about 10 to about 50 kg. Insome embodiments, manufacturing scale comprises a scale of about 33.4kg.

In some embodiments, an experiment is performed on a smaller scale togather information to be used to plan or perform synthesis on amanufacturing scale. In some embodiments, the results obtained on thesmaller scales are expected to be reproducible on manufacturing scale.In some embodiments, the results obtained on smaller scales are notexpected to be reproducible on manufacturing scale. In some embodiments,the yields obtained on manufacturing scale are greater than the yieldsobtained on smaller scales. In some embodiments, the yields obtained onmanufacturing scale are lesser than the yields obtained on smallerscales.

In one embodiment, a solution of a compound of Formula i in a solvent isprepared. A compound of Formula ii is then contacted to the solution,and the resultant mixture is heated in the presence of a base for aperiod of time sufficient to provide a compound of Formula iii. In someembodiments, the period of time is about 1 hour, about 2 hours, about 4hours, about 8 hours, about 12 hours, or about 24 hours. In someembodiments, the time is from about 1 hour to about 24 hours. In someembodiments, the base comprises lithium carbonate, sodium carbonate,potassium carbonate, sodium bicarbonate, a sodium phosphate, or apotassium phosphate. In some embodiments, the solvent comprises DMF. Insome embodiments, the temperature is about 50° C., about 70° C., about100° C., about 150° C., or a temperature effective to sustain refluxconditions. In some embodiments, the temperature is from about 50° C. toabout 200° C. The compound of Formula iii can be isolated from thereaction mixture and purified by any method known to one of skill in theart. Such methods include, but are not limited to, pouring an aqueousmixture into the reaction mixture, thereby effecting the precipitationof compound iii as a solid. The isolated compound of Formula iii mayoptionally be purified by any method known to one of skill in the art.Such methods include, but are not limited to, trituration with water.

In one embodiment, a solution of a compound of Formula iii in a solventis prepared, and the solution is contacted with a catalyst for a periodof time sufficient to provide a compound of Formula iv. In someembodiments, the period of time is about 1 hour, about 2 hours, about 4hours, about 8 hours, about 12 hours, or about 24 hours. In someembodiments, the time is from about 1 hour to about 24 hours. In someembodiments, the catalyst comprises palladium on carbon, platinum oncarbon, a transition metal salt, or a transition metal complex. In someembodiments, the solvent comprises N-methylpyrrolidone. In someembodiments, the temperature is about 50° C., about 70° C., about 100°C., about 150° C., about 190° C., about 200° C., or a temperatureeffective to sustain reflux conditions. In some embodiments, thetemperature is from about 50° C. to about 250° C. The compound ofFormula iv can be isolated from the reaction mixture and purified by anymethod known to one of skill in the art. Such methods include, but arenot limited to, in-line filtration. The isolated compound of Formula ivmay optionally be purified by any method known to one of skill in theart.

In one embodiment, a solution of a compound of Formula iv in a solventis prepared, and the solution is contacted with a base for a period oftime sufficient to provide a compound of Formula v (i.e., Compound (1)).In some embodiments, the period of time is about 1 hour, about 2 hours,about 4 hours, about 8 hours, about 12 hours, or about 24 hours. In someembodiments, the time is from about 1 hour to about 24 hours. In someembodiments, the base comprises lithium hydroxide, sodium hydroxide,potassium hydroxide, sodium methoxide, potassium methoxide, sodiumethoxide, potassium ethoxide, lithium carbonate, sodium carbonate,potassium carbonate, sodium bicarbonate, a sodium phosphate, or apotassium phosphate. In some embodiments, the solvent comprises water,methanol, ethanol, 2-propanol, t-butanol, or mixtures thereof. In someembodiments, the solvent comprises methanol and the base comprisessodium methoxide. In some embodiments, the temperature is about 35° C.,about 50° C., about 70° C., about 100° C., or a temperature effective tosustain reflux conditions. In some embodiments, the temperature is fromabout 25° C. to about 100° C. The compound of Formula v can be isolatedfrom the reaction mixture and purified by any method known to one ofskill in the art. Such methods include, but are not limited to,extraction. The isolated compound of Formula v may optionally bepurified by any method known to one of skill in the art. Such methodsinclude, but are not limited to, trituration.

Exemplary Pharmaceutical Compositions/Formulations

A pharmaceutical composition, as used herein, refers to a mixture of acompound of Formula I with other chemical components, such as carriers,stabilizers, diluents, dispersing agents, suspending agents, thickeningagents, and/or excipients. The pharmaceutical composition facilitatesadministration of the compound to an organism. Pharmaceuticalcomposition containing a compound of Formula I can be administered intherapeutically effective amounts as pharmaceutical compositions by anyconventional form and route known in the art including, but not limitedto: intravenous, oral, rectal, aerosol, parenteral, ophthalmic,pulmonary, transdermal, vaginal, otic, nasal, and topicaladministration.

One may administer the compound in a local rather than systemic manner,for example, via injection of the compound directly into an organ, oftenin a depot or sustained release formulation. Furthermore, one mayadminister pharmaceutical composition containing a compound of Formula Iin a targeted drug delivery system, for example, in a liposome coatedwith organ-specific antibody. The liposomes will be targeted to andtaken up selectively by the organ. In addition, the pharmaceuticalcomposition containing a compound of Formula I may be provided in theform of a rapid release formulation, in the form of an extended releaseformulation, or in the form of an intermediate release formulation. Insome embodiments, the extended release formulation releases the compoundfor over 1 hour, over 2 hours, over 3 hours, over 4 hours, over 6 hours,over 12 hours, over 24 hours, or more. In some embodiments, the extendedrelease formulation releases the compound at a steady rate for over 1hour, over 2 hours, over 3 hours, over 4 hours, over 6 hours, over 12hours, over 24 hours, or more.

For oral administration, a compound of Formula I can be formulatedreadily by combining the active compounds with pharmaceuticallyacceptable carriers or excipients well known in the art. Such carriersenable the compounds described herein to be formulated as tablets,powders, pills, dragees, capsules, liquids, gels, syrups, elixirs,slurries, suspensions and the like, for oral ingestion by a subject tobe treated. Generally, excipients such as fillers, disintegrants,glidants, surfactants, recrystallization inhibitors, lubricants,pigments, binders, flavoring agents, and so forth can be used forcustomary purposes and in typical amounts without affecting theproperties of the compositions.

Non-limiting examples of fillers include lactose monohydrate,microcrystalline cellulose, mannitol, xylitol, calcium diphosphate, andstarch.

Non-limiting examples of disintegrants include croscarmellose, sodiumstarch glycholate, crospovidone, sodium alginate, methyl cellulose, andcarboxymethyl cellulose sodium.

Non-limiting examples of glidants include magnesium stearate, colloidalsilicon dioxide, starch and talc.

Non-limiting examples of surfactants include sodium lauryl sulfate,sorbitan esters, poloxamers, PEG block copolymers, and polysorbates.

Non-limiting examples of recrystallization inhibitors include poloxamer188, poloxamer 407, Povidone K-90, or hypromellose.

Non-limiting examples of lubricants include magnesium stearate andcalcium stearate

Pharmaceutical preparations for oral use can be obtained by mixing oneor more solid excipient with one or more of the compounds describedherein, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Dragee cores are provided with suitablecoatings. For this purpose, concentrated sugar solutions may be used,which may optionally contain gum arabic, talc, polyvinylpyrrolidone,carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquersolutions, and suitable organic solvents or solvent mixtures. Dyestuffsor pigments may be added to the tablets or dragee coatings foridentification or to characterize different combinations of activecompound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. In someembodiments, the capsule comprises a hard gelatin capsule comprising oneor more of pharmaceutical, bovine, and plant gelatins. In certaininstances, a gelatin is alkaline processed. The push-fit capsules cancontain the active ingredients in admixture with filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for such administration.

For buccal or sublingual administration, the compositions may take theform of tablets, lozenges, or gels formulated in conventional manner.Parental injections may involve for bolus injection or continuousinfusion. The pharmaceutical composition of Compound (1) may be in aform suitable for parenteral injection as a sterile suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Pharmaceutical formulations for parenteral administrationinclude aqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compositions described herein can be administered topically and canbe formulated into a variety of topically administrable compositions,such as solutions, suspensions, lotions, gels, pastes, medicated sticks,balms, creams or ointments. Such pharmaceutical composition can containsolubilizers, stabilizers, tonicity enhancing agents, buffers andpreservatives.

Formulations suitable for transdermal administration of compounds havingthe structure of Formula (1) may employ transdermal delivery devices andtransdermal delivery patches and can be lipophilic emulsions orbuffered, aqueous solutions, dissolved and/or dispersed in a polymer oran adhesive. Such patches may be constructed for continuous, pulsatile,or on demand delivery of pharmaceutical agents. Still further,transdermal delivery of a compound of Formula I can be accomplished bymeans of iontophoretic patches and the like. Additionally, transdermalpatches can provide controlled delivery of a compound of Formula I. Therate of absorption can be slowed by using rate-controlling membranes orby trapping the compound within a polymer matrix or gel. Conversely,absorption enhancers can be used to increase absorption. An absorptionenhancer or carrier can include absorbable pharmaceutically acceptablesolvents to assist passage through the skin. For example, transdermaldevices are in the form of a bandage comprising a backing member, areservoir containing the compound optionally with carriers, optionally arate controlling barrier to deliver the compound to the skin of the hostat a controlled and predetermined rate over a prolonged period of time,and means to secure the device to the skin.

For administration by inhalation, the compositions of the presentinvention may be in a form as an aerosol, a mist or a powder.Pharmaceutical compositions of Formula (I) are conveniently delivered inthe form of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, such as, by way of example only, gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compound of Formula I may also be formulated in rectal compositionssuch as enemas, rectal gels, rectal foams, rectal aerosols,suppositories, jelly suppositories, or retention enemas, containingconventional suppository bases such as cocoa butter or other glycerides,as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and thelike. In suppository forms of the compositions, a low-melting wax suchas, but not limited to, a mixture of fatty acid glycerides, optionallyin combination with cocoa butter is first melted.

In practicing the methods of treatment or use provided herein,therapeutically effective amounts of a compound of Formula I providedherein are administered in a pharmaceutical composition to a mammalhaving a disease or condition to be treated. In some embodiments, themammal is a human. A therapeutically effective amount can vary widelydepending on the severity of the disease, the age and relative health ofthe subject, the potency of the compound used and other factors. Thecompounds can be used singly or in combination with one or moretherapeutic agents as components of mixtures.

Pharmaceutical compositions may be formulated in conventional mannerusing one or more physiologically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques, carriers, and excipients may be used assuitable and as understood in the art. Pharmaceutical compositionscomprising a compound of Formula (I) may be manufactured in aconventional manner, such as, by way of example only, by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or compression processes.

The pharmaceutical compositions can include at least onepharmaceutically acceptable carrier, diluent or excipient and a compoundof Formula (I) described herein as an active ingredient in free-baseform, or in a pharmaceutically acceptable salt form. In addition, themethods and pharmaceutical compositions described herein include the useof N-oxides, crystalline forms (also known as polymorphs), as well asactive metabolites of these compounds having the same type of activity.

Methods for the preparation of compositions comprising the compoundsdescribed herein include formulating the compounds with one or moreinert, pharmaceutically acceptable excipients or carriers to form asolid, semi-solid or liquid. Solid compositions include, but are notlimited to, powders, tablets, dispersible granules, capsules, cachets,and suppositories. Liquid compositions include solutions in which acompound is dissolved, emulsions comprising a compound, or a solutioncontaining liposomes, micelles, or nanoparticles comprising a compoundas disclosed herein. Semi-solid compositions include, but are notlimited to, gels, suspensions and creams. The compositions may be inliquid solutions or suspensions, solid forms suitable for solution orsuspension in a liquid prior to use, or as emulsions. These compositionsmay also contain minor amounts of nontoxic, auxiliary substances, suchas wetting or emulsifying agents, pH buffering agents, and so forth.

In some embodiments, the pharmaceutical composition is a soliddispersion delivery system.

In some embodiments, the solid dispersion delivery system compriseshydroxypropyl methylcellulose (HPMC).

In some embodiments, the solid dispersion delivery system compriseshydroxypropyl methylcellulose phthalate (HPMCP).

In some embodiments, the solid dispersion delivery system compriseshydroxypropyl methylcellulose acetate succinate (HPMCAS).

In some embodiments, the solid dispersion delivery system comprisesPoloxamer 188.

In some embodiments, the solid dispersion delivery system comprisesPoloxamer 407.

In some embodiments, the solid dispersion delivery system comprisesPovidone K-90.

In some embodiments, the pharmaceutical composition is a physicalmixture.

A summary of types of pharmaceutical compositions may be found, forexample, in Remington: The Science and Practice of Pharmacy, NineteenthEd (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N. Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999), each of which is incorporated by reference herein in itsentirety.

Spray Dried Compositions and Methods

In some embodiments, the present invention provides solid dispersioncompositions comprising a compound of Formula I:

or a pharmaceutically acceptable salt, N-oxide, active metabolite,prodrug, or solvate thereof; wherein R₁ is H or acetyl; R₂ is pyridyl orbenzimidazole; and a solid matrix. In some embodiments, the compound ofFormula I is dispersed in said solid matrix.

In some embodiments, the solid matrix is comprised of a polymer. In someembodiments, the polymer is a water soluble polymer. Non-limitingexamples of water soluble polymers used in solid dispersions includehydroxypropyl methyl cellulose (HPMC), polyvinylpyrrolidone (PVPblockcopolymers of ethylene oxide and propylene oxide ((K-25, 50 30, 90;PVP), hydroxypropyl cellulose (HPC), methyl cellulose (MC), andpolyethyleneglycol (PEG). In other embodiments, the polymer is solublein an aqueous solution. In particular embodiments, the polymer issoluble in an aqueous solution which has a pH of 5.5 or greater.Non-limiting examples of polymers soluble in aqueous solutions of pH 5.5or greater include sodium carboxymethylcellulose (NaCMC, sodiumcellulose glycolate) and hydroxypropylmethyl cellulose acetate succinate(HPMCAS). Other non-limiting examples of polymers suitable for use insolid dispersions include, e.g., of 3,4-dimethyl-phenomethylcarbamate(MPMC), hypromellose phthalate (HPMCP), Poloxamer 188, Poloxamer 407,Povidone K-90, poly(meth)acrylates (Eudragit), homopolymers ofN-vinyl-2-pyrrolidone, povidone, copovidone (Plasdone),carboxymethylethylcellulose (CMEC), cellulose acetate phthalate (CAP),methacrylic copolymer LD (L30 D55), methacrylic copolymer S (S-100),aminoalkyl methacrylate copolymer E (gastric coating base), poly(vinylacetal) diethylaminoacetate (AEA), ethylcellulose (EC), methacryliccopolymer RS (RS 30D), polyvinyl alcohol (PVA),hydroxypropylmethylcellulose (HPMC), HPMC 2208 (Metolose 90SH), HPMC2906 (Metolose 65SH), HPMC (Metolose 60SH), dextrin, pullulan, Acacia,tragacanth, sodium alginate, propylene glycol alginate, agar powder,gelatin, starch, processed starch, phospholipids, lecithin, glucomannan,polyethyleneglycol (PEG) cellulose acetate trimellitate (CAT),hydroxypropyl methyl cellulose acetate trimellitate (HPMCAT), andcarboxymethylcellulose acetate butyrate (CMCAB).

In some embodiments, the solid dispersion of the compound in matrix canbe prepared by forming a homogeneous solution or melt of the drug andpolymer, followed by solidifying the mixture, resulting in a solidcomposition of the compound dispersed in the solid matrix. In someembodiments, preparation of the solid dispersion comprises forming ahomogenous solution comprising the compound, the polymer, and a solvent,followed by solidifying the mixture by removal of the solvent. In someembodiments, the solvent is an organic solvent or a mixture of more thanone organic solvent. Non-limiting examples of organic solvents includedimethylformamide (DMF), acetone, methanol, ethanol, ethyl acetate,tetrahydrofuran, n-propanol, iso-propanol, butanol, methyl ethyl ketone,methyl iso-butyl ketone, propylacetate, acetonitrile, methylenechloride, toluene, 1,1,1-trichloroethane, dimethylacetamide, anddimethylsulfoxide. In particular embodiments, the solvent is methanol,ethanol, ethyl acetate, acetone, tetrahydrofuran, 2:1 acetone: methanol,2:1 methanol: tetrahydrofuran, 2:1 methanol: acetone, 6:1 DMF: water,14:7:2:1 acetone: methanol: DMF: water, 4:1:1 methanol: water: acetone,8:1 ethanol: water.

Methods for removing the solvent from the mixture are known in the art,and can include freeze-drying, vacuum drying, spray-drying, orcombinations thereof.

In particular embodiments, the solvent is removed by spray-drying. Theterm “spray-drying” generally broadly refers to atomizing the solutioninto a spray of small droplets and rapidly removing solvent from thedroplets using a spray-drying apparatus that facilitates rapidevaporation of solvent from the droplets. Spray-drying processes andspray-drying equipment are described generally in Perry's ChemicalEngineers' Handbook, pages 20-54 to 20-57 (Sixth Edition 1984). Solventevaporation can be facilitated by, e.g., maintaining the pressure in thespray-drying apparatus at a partial vacuum (for example, 0.01 to 0.50atm), contacting the droplets with a warm drying gas, or a combinationof these measures. In some embodiments, spray drying comprisescontacting the spray of droplets with a drying gas.

In some embodiments, removal of the solvent by spray drying results insolid dispersion compositions in the form of particles. The particlescan have a mean diameter of about 100 μm or less, about 95 μm or less,about 90 μm or less, about 85 μm or less, about 80 μm or less, about 75μm or less, about 70 μm or less, about 65 μm or less, about 60 μm orless, about 55 μm or less, about 50 μm or less, about 45 μm or less,about 40 μm or less, about 35 μm or less, about 30 μm or less, about 25μm or less, or about 20 μm or less. In some embodiments, the particleshave a mean diameter of about 50-100 μm, about 30-75 μm, about 25-50 μm,about 20-30 μm, about 10-25 μm, or about 15-20 μm. Particle size can bemeasured using particle size measuring techniques known to those ofskill in the art. Non-limiting examples of particle size measuringtechniques include sedimentation field flow fractionation, photoncorrelation spectroscopy, laser diffraction or disk centrifugation.Another useful characteristic diameter of the droplets produced by anatomizer is D90, the droplet diameter corresponding to the diameter ofdroplets that make up 90% of the total liquid volume. In someembodiments, the particles of the composition have diameters spanningabout 10-20 μm at D90, 15-20 μm at D90, or 17-19 μm at D90.

In some embodiments, spray-drying results in compositions in which thecompound of Formula I is amorphous. Methods and characterization ofamorphousness are described herein. Exemplary Methods of Administrationand Treatment Methods

Compositions comprising a compound of Formula I-III can be used in thepreparation of medicaments for the treatment of diseases or conditionsin which steroid hormone nuclear receptor activity contributes to thepathology and/or symptoms of the disease. In addition, a method fortreating any of the diseases or conditions described herein in a subjectin need of such treatment, involves administration of pharmaceuticalcompositions containing at least one compound of Formula (1), or apharmaceutically acceptable salt, pharmaceutically acceptable N-oxide,pharmaceutically active metabolite, pharmaceutically-acceptable prodrug,or pharmaceutically acceptable solvate thereof, intherapeutically-effective amounts to said subject.

The compositions containing the compound(s) described herein can beadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, the compositions are administered to a subjectalready suffering from a disease or condition, in an amount sufficientto cure or at least partially arrest the symptoms of the disease orcondition, or to cure, heal, improve, or ameliorate the conditionitself. Amounts effective for this use will depend on the severity andcourse of the disease or condition, previous therapy, the subject'shealth status, weight, and response to the drugs, and the judgment ofthe treating physician.

Once improvement of the subject's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease or condition isretained. Subjects can, however, require intermittent treatment on along-term basis upon any recurrence of symptoms.

In certain instances, it may be appropriate to administertherapeutically effective amounts of at least one of the compoundsdescribed herein (or a pharmaceutically acceptable salts,pharmaceutically-acceptable N-oxides, pharmaceutically activemetabolites, pharmaceutically-acceptable prodrugs, and pharmaceuticallyacceptable solvates thereof) in combination with another therapeuticagent. By way of example only, if one of the side effects experienced bya subject upon receiving one of the compounds herein is inflammation,then it may be appropriate to administer an anti-inflammatory agent incombination with the initial therapeutic agent. Or, by way of exampleonly, the therapeutic effectiveness of one of the compounds describedherein may be enhanced by administration of an adjuvant (i.e., by itselfthe adjuvant may only have minimal therapeutic benefit, but incombination with another therapeutic agent, the overall therapeuticbenefit to the subject is enhanced). Or, by way of example only, thebenefit of experienced by a subject may be increased by administeringone of the compounds described herein with another therapeutic agent(which also includes a therapeutic regimen) that also has therapeuticbenefit. In any case, regardless of the disease or condition beingtreated, the overall benefit experienced by the subject may simply beadditive of the two therapeutic agents or the subject may experience asynergistic benefit. Where the compounds described herein areadministered in conjunction with other therapies, dosages of theco-administered compounds will of course vary depending on the type ofco-drug employed, on the specific drug employed, on the disease orcondition being treated and so forth. In addition, when co-administeredwith one or more biologically active agents, the compound providedherein may be administered either simultaneously with the biologicallyactive agent(s), or sequentially. If administered sequentially, theattending physician will decide on the appropriate sequence ofadministering protein in combination with the biologically activeagent(s).

In any case, the multiple therapeutic agents (one of which is one of thecompounds described herein) may be administered in any order or evensimultaneously. If simultaneously, the multiple therapeutic agents maybe provided in a single, unified form, or in multiple forms (by way ofexample only, either as a single pill or as two separate pills). One ofthe therapeutic agents may be given in multiple doses, or both may begiven as multiple doses. If not simultaneous, the timing between themultiple doses may vary from more than zero weeks to less than fourweeks. In addition, the combination methods, compositions andformulations are not to be limited to the use of only two agents.Multiple therapeutic combinations are envisioned.

In addition, compounds of Formula I-III may also be used in combinationwith procedures that may provide additional or synergistic benefit tothe subject. By way of example only, subjects are expected to findtherapeutic and/or prophylactic benefit in the methods described herein,wherein pharmaceutical composition of Formula (I) and/or combinationswith other therapeutics are combined with genetic testing to determinewhether that individual is a carrier of a mutant gene that is known tobe correlated with certain diseases or conditions.

Compounds of Formula I-III and combination therapies can be administeredbefore, during or after the occurrence of a disease or condition, andthe timing of administering the composition containing a compound canvary. Thus, for example, the compounds can be used as a prophylactic andcan be administered continuously to subjects with a propensity toconditions or diseases in order to prevent the occurrence of the diseaseor condition. The compounds and compositions can be administered to asubject during or as soon as possible after the onset of the symptoms.The administration of the compounds can be initiated within the first 48hours of the onset of the symptoms, preferably within the first 48 hoursof the onset of the symptoms, more preferably within the first 6 hoursof the onset of the symptoms, and most preferably within 3 hours of theonset of the symptoms. The initial administration can be via any routepractical, such as, for example, an intravenous injection, a bolusinjection, infusion over 5 minutes to about 5 hours, a pill, a capsule,transdermal patch, buccal delivery, and the like, or combinationthereof. A compound is preferably administered as soon as is practicableafter the onset of a disease or condition is detected or suspected, andfor a length of time necessary for the treatment of the disease, suchas, for example, from about 1 month to about 3 months. The length oftreatment can vary for each subject, and the length can be determinedusing the known criteria. For example, the compound or a formulationcontaining the compound can be administered for at least 2 weeks,preferably about 1 month to about 3 years and in some embodiments fromabout 1 month to about 10 years. In other embodiments, the compound isadministered once a day from 90 days to 2 years.

The pharmaceutical composition described herein may be in unit dosageforms suitable for single administration of precise dosages. In unitdosage form, the formulation is divided into unit doses containingappropriate quantities of one or more compounds. The unit dosage may bein the form of a package containing discrete quantities of theformulation. Non-limiting examples are packaged tablets or capsules, andpowders in vials or ampoules. Aqueous suspension compositions can bepackaged in single-dose non-reclosable containers. Alternatively,multiple-dose reclosable containers can be used, in which case it istypical to include a preservative in the composition. By way of exampleonly, formulations for parenteral injection may be presented in unitdosage form, which include, but are not limited to ampoules, or inmulti-dose containers, with an added preservative.

The daily dosages appropriate for any of the compounds described hereinare from about 0.03 to 60 mg/kg per body weight. An indicated dailydosage in a larger mammal, including, but not limited to, humans, is inthe range from about 1 mg to about 4000 mg, conveniently administered inone or more doses, including, but not limited to, up to five times a dayor in retard form. Suitable unit dosage forms for oral administrationcomprise from about 1 mg to about 4000 mg active ingredient. In someembodiments, a single dose of compounds of Formula (1) is within therange of about 50 mg to about 3500 mg. In some embodiments, a singledose of compounds of Formula (1) is about 90 mg, about 200 mg, about 250mg, about 325 mg, about 500 mg, about 650 mg, about 975 mg, about 1300mg, about 1625 mg, about 1950 mg, about 2600 mg or about 3250 mg. Insome embodiments, an administration of compounds of Formula (1) of about90 mg, about 325 mg, about 500 mg, about 650 mg, about 975 mg, about1300 mg, about 1625 mg, about 1950 mg, about 2600 mg or about 3250 mg isgiven as multiple doses.

In some embodiments, the single dose of compounds of Formula (a) isbetween 90 to 3500 mgs and the compound is administered to a subject forbetween 90 days to two years.

Such dosages may be altered depending on a number of variables, notlimited to the activity of the compound used, the disease or conditionto be treated, the mode of administration, the requirements of theindividual subject, the severity of the disease or condition beingtreated, and the judgment of the practitioner.

Exemplary Methods of Providing Therapy

The present invention provides therapeutic strategies for the treatmentof cancer or other disease in subjects. In some embodiments, the diseaseis polycystic ovarian disease. In some embodiments, the cancer inprostate cancer. In other embodiments, the cancer is breast cancer. Inyet other embodiments, the cancer is ovarian cancer. In someembodiments, the subject is human. In other embodiments, the subject isnot a human.

In particular embodiments, the present invention provides preparationsand regimens for the use of a compound of Formula I or formula II in thetreatment of prostate cancer. In some embodiments, the prostate canceris castration resistance prostate cancer. In some embodiments, theprostate cancer is chemotherapy naïve prostate cancer.

In some embodiments, the present invention provides therapeutic regimensthat involve oral administration of a compound of Formula I or formulaII.

In some embodiments, the present invention provides therapeutic regimensthat involve administration of multiple doses of a compound of Formula Ior formula II. In some embodiments, different doses are spaced apart intime. In some embodiments, all doses contain the same amount of acompound of Formula I or formula II. In some embodiments, differentdoses contain different amounts of a compound of Formula I or formulaII. In some embodiments, different doses that are separated in time areseparated from one another by the same amount of time; in someembodiments, different doses that are separated in time are separatedfrom one another by different amounts of time. In some embodiments, thepresent invention provides dosing regimens that include administrationof a plurality of doses separated by a regular time interval (orintervals), followed by a rest period, optionally followed by a secondplurality of doses separated by a regular time interval (or intervals).

In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168 or more doses of acompound of Formula I or formula II are administered. In someembodiments, at least 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91,98, 105, 112, 119, 126, 133, 140, 147, 154, 161, 168, or more doses of acompound of Formula I or formula II are administered.

EXAMPLES

Galeterone is a novel drug that exhibits three mechanisms of action toinhibit AR activity, via inhibition of de novo androgen synthesis,blocking the ligand binding domain to prevent androgen binding, andinducing AR degradation. Thus, in this study we evaluated therelationship between the mechanism of action of this drug and theutility of various biomarkers, including the status of androgen receptorin the tumor being treated.

Example 1 Galeterone Downregulates Both Wild-Type and Mutant AR

An experiment to detect expression of full-length AR and AR-V7 variantproteins was conducted in a CWR22rv1 cell line which constitutivelyexpresses both AR and AR-V7. As shown in, FIG. 2, galeteronedownregulates full-length and splice variant AR and reduces cellproliferation in this cell line. See also FIG. 4. Further, galeteronesuccessfully overcomes abiraterone and enzalutamide resistance due to ARsplice variants (FIG. 3). Galeterone, but not enzalutamide, reducesfull-length and splice variant AR-V7 protein. Further, galeteronereduces AR-V7 in DU145 cells transfected with AR-V7 splice variant (FIG.5). Similarly, lower levels of galeterone reduce full-length and splicevariant AR-V7 with 72 hour exposure (FIG. 6).

Example 2 Galeterone is Effective in a Model of Enzalutamide Resistance

As a pre-clinical model of Enzalutamide resistance, drug resistant andCRPC cell lines were derived from three generations of serially passagedEnzalutamide resistant, or vehicle control treated, LNCaP xenografts.Resistant cells (“49C” and “49F”) were maintained in vitro underconstant exposure to 10 μM of Enzalutamide and were used to study theanti-cancer and AR targeting effects of Galeterone in the Enzalutamideresistant setting. Both 49F and 49C cell lines were found to have lowexpression of AR-V7. Using crystal violet and MTI assays, we found thatGaleterone had anti-proliferative effects in LNCaP cells, in CRPC cells,and most importantly, in those resistant to Enzalutamide (FIG. 7).Dose-response studies of Galeterone in LNCaP cells and Enzalutamideresistant cell lines demonstrated similar EC50 for Galeterone inreducing cell viability in Enzalutamide resistant cell lines 49F and49C, as compared to the EC50 for Galeterone in Enzalutamide responsiveLNCaP cells (FIG. 8). Enzalutamide treatment reduced AR and PSA proteinexpression levels in LNCaP cells but not in the Enzalutamide resistantcell line (FIG. 9A). Compared to Enzalutamide treatment, Galeteroneinduced a greater reduction in AR and prostate-specific antigen (PSA)protein expression (FIG. 9B). Strikingly, the effects of Galeterone werestill observed in resistant cells (FIG. 9B), which show no decrease inAR protein expression or PSA protein expression (FIG. 9A). To determinethe effects of Enzalutamide and Galeterone on AR nuclear translocation,LNCaP and Enzalutamide resistant cell lines were treated with thesynthetic androgen R1881 and/or Enzalutamide. AR localization wasvisualized by immunocytochemistry. R1881 alone caused robust ARtranslocation to the nucleus in both LNCaP and Enzalutamide resistantcell lines (FIGS. 10-13) Co-treatment with R1881 and enzalutamidereduced AR nuclear translocation as compared to R1881 treatment alone inLNCaP cells but not Enzalutamide resistant cells (FIGS. 10-13). Todetermine the effects of enzalutamide and Galeterone on AR activity, anAR luciferase assay was performed in the enzalutamide-responsive CPRCcell line and the Enzalutamide resistant cell lines 49C and 49F. Allcell lines, untreated, exhibited high AR activity levels (FIG. 14).Enzalutamide treatment reduced AR activity in theenzalutamide-responsive cell line but not in the resistant cell lines49C or 49F (FIG. 14). Galeterone, by contrast, reduced AR activity inall three cell lines (FIG. 14). FIG. 15A shows the design of animmunofluorescence experiment to visualize nuclear localization of testcompounds used, and FIG. 15B shows that galeterone, but notenzalutamide, reduces AR nuclear translocation (as seen by increasedgreen cytoplasmic staining vs. control or enzalutamide and less nucleargreen staining). Together, the data show that Galeterone stronglyinhibits AR activity and suppresses castration-resistant LNCaP growth aswell as enzalutamide-resistant cell growth in vitro.

Example 3 Galterone Activity in Castration Resistant Xenograft Tumorswhich Express Mutated Androgen Receptor

Galeterone was tested in a xenograft model of CRPC in mice. FIG. 16shows that castration resistant tumors which express AR-V7 respond togaleterone. AR-V7 was detected in LuCaP136 castration resistantxenograft tumors using RT-PCR.

Example 4 Galeterone Downregulates Mutant Androgen Receptors

AR point mutation AR-T878A is commonly present in hormone refractorytumors and because the mutation is in the androgen binding site themutant AR binds to steroids and drugs very differently. In particular,the AR-T878A mutant binds to progesterone which is elevated inabiraterone-treatment thus tumors expressing this mutation are resistantto abiraterone. An experiment was conducted to FIG. 17 shows thatgaleterone downregulates androgen receptors carrying the AR-T878Amutation. This effect is seen on either LNCaP cells or AR negative PC3cells that have been transfected with AR-T878A.

Example 5 CRPC Patients Having Androgen Receptor Splice Variants Respondto Galeterone Therapy

Galeterone was administered to a group of patients for 12 weeks at 2550mg daily. A group of six patients naive to previous CRPC therapy wasanalyzed with respect to AR status. Four of the six patients wereidentified as having AR receptors with a C-terminal loss as determinedby the evaluation of C-terminal androgen receptor expression in relationto N-terminal androgen receptor expression. The results of theexperiments are shown in FIG. 22. All four of the patients having thisAR variant had maximal reductions in PSA levels of at least 50%. Theother two patients, which did not have AR receptors with a loss of theC-terminal domain, did not respond as strongly to galeterone asevidenced by the lesser reduction in PSA.

Therefore, galeterone is a potent inhibitor of the AR pathway and mayrepresent the next generation of hormone therapy for patients with notonly CRPC but also Enzalutamide resistant disease. Furthermore, asgaleterone is a potent inhibitor of the AR pathway, it may represent analternative to abiraterone or to patients who are resistant toabiraterone therapy. While preferred embodiments of the presentinvention have been shown and described herein, it will be obvious tothose skilled in the art that such embodiments are provided by way ofexample only. Numerous variations, changes, and substitutions will nowoccur to those skilled in the art without departing from the invention.It should be understood that various alternatives to the embodiments ofthe invention described herein may be employed in practicing theinvention. It is intended that the following claims define the scope ofthe invention and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

Example 6 Detection of Mutated AR by Amplification

Mutated AR in patient samples may be detected by PCR. Quantitativereal-time PCR methods are known in the literature, specifically asdescribed in Luo et al., US2011/0110926. As described Cancer Research2009, 69:16-22, for RT-PCR analyses, total RNA was isolated and reversetranscribed to form cDNA and was used in a RT-PCR analysis. PCR primerswere designed as described to specifically amplify transcript sequencesthat are known in the NH2 terminal (5′ primers) (for example primerP6/P7/P9) and within the mutated forms (truncated, for example P7) ofthe AR protein (i.e. specific to AR-V7) mRNA and can be readily detectedwithin about 30 (specifically 28) PCR amplification cycles. Usingmethods as described it would be possible to detect expression levels oftruncated AR in samples from patients with prostate cancer. In thisanalysis, and due to variable expression levels in patient samples, thegene SF3A3, was used as a reference gene for normalization.

1-63. (canceled)
 64. A method comprising steps of: administering therapyto a subject having prostate cancer and expressing ARV-7, which therapycomprises administering a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein R₁ is H and R₂ isbenzamidazole.
 65. The method of claim 64, wherein the ARV-7 has beendetected in a sample from the subject.
 66. The method of claim 65,wherein the sample is selected from the group consisting of whole blood,serum, plasma, tissue, semen, cell, biopsy, mucous, feces, bone, teeth,nasal or throat or cheek swab, urine, skin, tears, organ biopsy, tumorbiopsy or tumor tissue, circulating tumor cells, exosomes from a primarytumor or from metastatic tissue, and combinations thereof.
 67. Themethod of claim 65, wherein the sample is a processed sample.
 68. Themethod of claim 67, wherein the sample is a processed whole bloodsample.
 69. The method of claim 65, wherein the sample is a tumor tissuesample or tumor biopsy sample.
 70. The method of claim 64, wherein thesample is a sample enriched for circulating tumor cells.
 71. The methodof claim 65, where the sample is a sample enriched for circulating DNA.72. The method of claim 64, wherein the ARV-7 is expressed incirculating tumor cells (CTC).
 73. The method of claim 64, wherein theprostate cancer is resistant to castration.
 74. The method of claim 64,wherein the subject has undergone chemical castration or surgicalcastration.
 75. The method of claim 64, wherein the prostate cancer isresistant to an androgen receptor antagonist.
 76. The method of claim75, wherein the androgen receptor antagonist is enzalutamide orbicalutamide or ARN-509.
 77. The method of claim 64, wherein the canceris resistant to a CYP17 inhibitor.
 78. The method of claim 77, whereinthe CYP17 inhibitor is abiraterone.
 79. The method of claim 64, whereinthe cancer is resistant to a taxane.
 80. The method of claim 79, whereinthe taxane is docetaxel or cabazitaxel.
 81. The method of claim 64,wherein the cancer is androgen dependent.
 82. The method of claim 64,wherein the step of administering comprises administering a daily dosagewithin a range of about 0.03 to about 60 mg/kg body weight.
 83. Themethod of claim 64, wherein the step of administering comprisesadministering about 2,550 mg daily.
 84. The method of claim 64, whereinthe step of administering comprises administering at least one unitdosage form comprising from about 90 mg to about 3,500 mg activeingredient.
 85. The method of claim 64, wherein the step ofadministering comprises administering orally.
 86. The method of claim64, wherein the step of administering comprises administering for aperiod of time sufficient that the subject shows one or more of PSAdecline relative to pretreatment level, pain improvement, quality oflife improvement as assessed by direct patient measure, change in CTCcount, change in CTC characterization, PSA progression free survival,radiographic progression free survival, and induction of immunity totumor antigens.
 87. The method of claim 64, wherein the step ofadministering comprises administering for a period of time sufficientthat the subject shows one or more of: decrease in numbers of CTC,increase in apoptotic CTC, decrease in PSA or reduction in PSA doublingtime, increase in PSMA expression as determined by imaging modalitiessuch as radio labeled ligands of PSMA or antibodies than bind PSMA,reduction in tumor 18F-DHT-PET signal, a tissue biopsy test thatdifferentiates indolent from aggressive disease in formalin-fixed,paraffin-embedded tissue samples, inhibition of androgen receptortagging, and removal of androgen receptor from cells.
 88. The method ofclaim 86, wherein the PSA decline is a maximal reduction in PSA level ofat least 50%.
 89. The method of claim 86, wherein the radiographicprogression free survival is evaluated using a radiolabeled ligand. 90.The method of claim 89, wherein the radiolabeled ligand is aradiolabeled ligand of prostate specific membrane antigen (PSMA). 91.The method of claim 64, wherein the step of administering comprisesadministering to a population of subjects with prostate cancer andexpressing ARV-7 and is effective to achieve one or more of PSA declinerelative to pretreatment level, pain improvement, quality of lifeimprovement as assessed by direct patient measure, change in CTC count,change in CTC characterization, PSA progression free survival,radiographic progression free survival, and induction of immunity totumor antigens in the population.